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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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Wnt target genes are down-regulated in Sox2-expressing osteoblasts. (A) Level of expression of Wnt target genes in Sox2#16 osteoblasts normalized to OB1. These data are derived from the same microarray analysis described in Table I. Normalizations were done as described in Materials and methods. (B) Real-time PCR analysis of Wnt target genes in osteoblast-expressing Sox2. Total RNA from OB1, OB1/vector, and OB1/Sox2#16 cells was reverse transcribed amplified by real-time PCR using SYBR green I for detection. For each PCR reaction, crosspoint values for engrailed1 and connexin 43 were normalized using β-catenin values, and relative expression level obtained for OB1 osteoblast (black bar) was set as 100%. Each bar represents the mean of three independent experiments ± SD.
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fig5: Wnt target genes are down-regulated in Sox2-expressing osteoblasts. (A) Level of expression of Wnt target genes in Sox2#16 osteoblasts normalized to OB1. These data are derived from the same microarray analysis described in Table I. Normalizations were done as described in Materials and methods. (B) Real-time PCR analysis of Wnt target genes in osteoblast-expressing Sox2. Total RNA from OB1, OB1/vector, and OB1/Sox2#16 cells was reverse transcribed amplified by real-time PCR using SYBR green I for detection. For each PCR reaction, crosspoint values for engrailed1 and connexin 43 were normalized using β-catenin values, and relative expression level obtained for OB1 osteoblast (black bar) was set as 100%. Each bar represents the mean of three independent experiments ± SD.

Mentions: Therefore, we used the microarray analysis described in the previous section to examine the expression of Wnt target genes in Sox2#16 cells. Fig. 5 A shows that out of 18 Wnt target genes in Fig. 1 A that were down-regulated in AP or CR cells, 12 were also down-regulated in the Sox2#16 cells. It is interesting to note that the expression of the Wnt target gene fra-1, which was up-regulated in the AP or CR cells because this gene is a direct FGF target, was unchanged in the Sox2-expressing osteoblasts, whereas the Wnt targets jun and twist, which were unchanged in AP and CR cells, are down-regulated in Sox2#16 cells (Fig. 5 A). Some of the Wnt target genes whose expression is not changed in Sox2#16 cells were also not strongly down-regulated in the AP and CR cells (BMP-4 and noggin). The validity of the microarray data was verified by performing real-time RT-PCR of a few strongly down-regulated Wnt target genes (Fig. 5 B). Thus, constitutive Sox2 expression causes down-regulation of the expression of a large number of Wnt target genes, while inhibiting osteoblast differentiation.


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)

Wnt target genes are down-regulated in Sox2-expressing osteoblasts. (A) Level of expression of Wnt target genes in Sox2#16 osteoblasts normalized to OB1. These data are derived from the same microarray analysis described in Table I. Normalizations were done as described in Materials and methods. (B) Real-time PCR analysis of Wnt target genes in osteoblast-expressing Sox2. Total RNA from OB1, OB1/vector, and OB1/Sox2#16 cells was reverse transcribed amplified by real-time PCR using SYBR green I for detection. For each PCR reaction, crosspoint values for engrailed1 and connexin 43 were normalized using β-catenin values, and relative expression level obtained for OB1 osteoblast (black bar) was set as 100%. Each bar represents the mean of three independent experiments ± SD.
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Related In: Results  -  Collection

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fig5: Wnt target genes are down-regulated in Sox2-expressing osteoblasts. (A) Level of expression of Wnt target genes in Sox2#16 osteoblasts normalized to OB1. These data are derived from the same microarray analysis described in Table I. Normalizations were done as described in Materials and methods. (B) Real-time PCR analysis of Wnt target genes in osteoblast-expressing Sox2. Total RNA from OB1, OB1/vector, and OB1/Sox2#16 cells was reverse transcribed amplified by real-time PCR using SYBR green I for detection. For each PCR reaction, crosspoint values for engrailed1 and connexin 43 were normalized using β-catenin values, and relative expression level obtained for OB1 osteoblast (black bar) was set as 100%. Each bar represents the mean of three independent experiments ± SD.
Mentions: Therefore, we used the microarray analysis described in the previous section to examine the expression of Wnt target genes in Sox2#16 cells. Fig. 5 A shows that out of 18 Wnt target genes in Fig. 1 A that were down-regulated in AP or CR cells, 12 were also down-regulated in the Sox2#16 cells. It is interesting to note that the expression of the Wnt target gene fra-1, which was up-regulated in the AP or CR cells because this gene is a direct FGF target, was unchanged in the Sox2-expressing osteoblasts, whereas the Wnt targets jun and twist, which were unchanged in AP and CR cells, are down-regulated in Sox2#16 cells (Fig. 5 A). Some of the Wnt target genes whose expression is not changed in Sox2#16 cells were also not strongly down-regulated in the AP and CR cells (BMP-4 and noggin). The validity of the microarray data was verified by performing real-time RT-PCR of a few strongly down-regulated Wnt target genes (Fig. 5 B). Thus, constitutive Sox2 expression causes down-regulation of the expression of a large number of Wnt target genes, while inhibiting osteoblast differentiation.

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