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Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta.

Grafe I, Yang T, Alexander S, Homan EP, Lietman C, Jiang MM, Bertin T, Munivez E, Chen Y, Dawson B, Ishikawa Y, Weis MA, Sampath TK, Ambrose C, Eyre D, Bächinger HP, Lee B - Nat. Med. (2014)

Bottom Line: Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms.In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity.Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

ABSTRACT
Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap(-/-)) and dominant (Col1a2(tm1.1Mcbr)) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap(-/-) mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity. Anti-TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap(-/-) mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

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Inhibition of upregulated TGFβ signaling improves the bone phenotype in a mouse model of dominant OI (Col1a2tm1.1Mcbr). (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Col1a2tm1.1Mcbr mice. Results are shown as fold change of the mean of WT group±SD; n=3 per group. (b) Western blot analysis showing activated Smad2 (pSmad2) relative to total Smad2 protein in P3 calvaria of WT and Col1a2tm1.1Mcbr mice; n=3 per group. (c) Quantification of the Western blot seen in b. Results are shown as fold change of the mean of WT group±SD. (d) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Col1a2tm1.1Mcbr and 1D11-treated Col1a2tm1.1Mcbr mice after treatment for 8 weeks (scale bar=500 μm). (e) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and thickness (Tb.Th) in WT, control Col1a2tm1.1Mcbr and 1D11 treated Col1a2tm1.1Mcbr mice. Results are shown as means±SDs, n=6 per group. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.
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Figure 4: Inhibition of upregulated TGFβ signaling improves the bone phenotype in a mouse model of dominant OI (Col1a2tm1.1Mcbr). (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Col1a2tm1.1Mcbr mice. Results are shown as fold change of the mean of WT group±SD; n=3 per group. (b) Western blot analysis showing activated Smad2 (pSmad2) relative to total Smad2 protein in P3 calvaria of WT and Col1a2tm1.1Mcbr mice; n=3 per group. (c) Quantification of the Western blot seen in b. Results are shown as fold change of the mean of WT group±SD. (d) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Col1a2tm1.1Mcbr and 1D11-treated Col1a2tm1.1Mcbr mice after treatment for 8 weeks (scale bar=500 μm). (e) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and thickness (Tb.Th) in WT, control Col1a2tm1.1Mcbr and 1D11 treated Col1a2tm1.1Mcbr mice. Results are shown as means±SDs, n=6 per group. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.

Mentions: Because of the clinical overlap of some recessive and dominant forms of OI, it is possible that dysregulation of TGFβ signaling is a common disease mechanism. To address this hypothesis, we investigated the status of TGFβ signaling in a mouse model of dominant OI. Knock-in mice carrying a G610C mutation in the Col1a2 gene (Col1a2tm1.1Mcbr) phenocopy a dominantly inherited, moderate form of OI that was identified in an Amish population. Compared with bone samples of WT mice, we found higher expression of the TGFβ target genes Cdkn1a and Serpine1 in Col1a2tm1.1Mcbr mice, indicating upregulation of TGFβ signaling (Fig. 4a). Consistently, immunoblot analyses showed a greater ratio of pSmad2/total Smad2 in bone of Col1a2tm1.1Mcb compared with WT mice, similar to our observation in Crtap−/− mice (Fig. 4b,c).


Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta.

Grafe I, Yang T, Alexander S, Homan EP, Lietman C, Jiang MM, Bertin T, Munivez E, Chen Y, Dawson B, Ishikawa Y, Weis MA, Sampath TK, Ambrose C, Eyre D, Bächinger HP, Lee B - Nat. Med. (2014)

Inhibition of upregulated TGFβ signaling improves the bone phenotype in a mouse model of dominant OI (Col1a2tm1.1Mcbr). (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Col1a2tm1.1Mcbr mice. Results are shown as fold change of the mean of WT group±SD; n=3 per group. (b) Western blot analysis showing activated Smad2 (pSmad2) relative to total Smad2 protein in P3 calvaria of WT and Col1a2tm1.1Mcbr mice; n=3 per group. (c) Quantification of the Western blot seen in b. Results are shown as fold change of the mean of WT group±SD. (d) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Col1a2tm1.1Mcbr and 1D11-treated Col1a2tm1.1Mcbr mice after treatment for 8 weeks (scale bar=500 μm). (e) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and thickness (Tb.Th) in WT, control Col1a2tm1.1Mcbr and 1D11 treated Col1a2tm1.1Mcbr mice. Results are shown as means±SDs, n=6 per group. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.
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Figure 4: Inhibition of upregulated TGFβ signaling improves the bone phenotype in a mouse model of dominant OI (Col1a2tm1.1Mcbr). (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Col1a2tm1.1Mcbr mice. Results are shown as fold change of the mean of WT group±SD; n=3 per group. (b) Western blot analysis showing activated Smad2 (pSmad2) relative to total Smad2 protein in P3 calvaria of WT and Col1a2tm1.1Mcbr mice; n=3 per group. (c) Quantification of the Western blot seen in b. Results are shown as fold change of the mean of WT group±SD. (d) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Col1a2tm1.1Mcbr and 1D11-treated Col1a2tm1.1Mcbr mice after treatment for 8 weeks (scale bar=500 μm). (e) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and thickness (Tb.Th) in WT, control Col1a2tm1.1Mcbr and 1D11 treated Col1a2tm1.1Mcbr mice. Results are shown as means±SDs, n=6 per group. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.
Mentions: Because of the clinical overlap of some recessive and dominant forms of OI, it is possible that dysregulation of TGFβ signaling is a common disease mechanism. To address this hypothesis, we investigated the status of TGFβ signaling in a mouse model of dominant OI. Knock-in mice carrying a G610C mutation in the Col1a2 gene (Col1a2tm1.1Mcbr) phenocopy a dominantly inherited, moderate form of OI that was identified in an Amish population. Compared with bone samples of WT mice, we found higher expression of the TGFβ target genes Cdkn1a and Serpine1 in Col1a2tm1.1Mcbr mice, indicating upregulation of TGFβ signaling (Fig. 4a). Consistently, immunoblot analyses showed a greater ratio of pSmad2/total Smad2 in bone of Col1a2tm1.1Mcb compared with WT mice, similar to our observation in Crtap−/− mice (Fig. 4b,c).

Bottom Line: Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms.In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity.Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

ABSTRACT
Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap(-/-)) and dominant (Col1a2(tm1.1Mcbr)) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap(-/-) mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity. Anti-TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap(-/-) mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

Show MeSH
Related in: MedlinePlus