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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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Phenotypic correction of Crtap−/− mice after treatment with the TGFβ neutralizing antibody 1D11. (a) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks (scale bar=500μm). (b) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=8 per group. (c) Histomorphometric analysis of L4 for osteoclast (N.Oc/BS) and osteoblast (N.Ob/BS) numbers per bone surface, and numbers of osteocytes per bone area (N.Ot/B.Ar) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=6 per group. (d) Hematoxylin/eosin staining of inflated lungs of 16-week-old wildtype (WT), control Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks. Representative images of n=8 mice per group are shown (scale bar=100 μm). (e) Quantification of the distance between alveolar structures by the mean-linear-intercept (MLI) method in lungs of WT, control Crtap−/− and 1D11-treated Crtap−/− mice. Results are shown as means±SDs, n=8 mice per group, 10 images analyzed per mouse. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.
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Figure 2: Phenotypic correction of Crtap−/− mice after treatment with the TGFβ neutralizing antibody 1D11. (a) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks (scale bar=500μm). (b) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=8 per group. (c) Histomorphometric analysis of L4 for osteoclast (N.Oc/BS) and osteoblast (N.Ob/BS) numbers per bone surface, and numbers of osteocytes per bone area (N.Ot/B.Ar) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=6 per group. (d) Hematoxylin/eosin staining of inflated lungs of 16-week-old wildtype (WT), control Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks. Representative images of n=8 mice per group are shown (scale bar=100 μm). (e) Quantification of the distance between alveolar structures by the mean-linear-intercept (MLI) method in lungs of WT, control Crtap−/− and 1D11-treated Crtap−/− mice. Results are shown as means±SDs, n=8 mice per group, 10 images analyzed per mouse. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.

Mentions: To understand whether upregulated TGFβ signaling represents a causal mechanism contributing to the bone and lung phenotypes in Crtap−/− mice, we performed a rescue experiment with a pan-TGFβ neutralizing antibody (1D11). Eight week old Crtap−/− mice were treated with 1D11 for eight weeks; control Crtap−/− and WT mice received a non-specific control antibody (13C4). 1D11 did not change body weight of the treated Crtap−/− mice, indicating that TGFβ inhibition did not affect their general nutritional status (Supplementary Fig. 1). Mass spectrometric and collagen cross-links analyses showed that 1D11 did not considerably change the status of P986 3-hydroxylation or collagen crosslinks in Crtap−/− mice, suggesting that dysregulated TGFβ signaling is a consequence of the altered collagen, and not directly involved in intracellular collagen processing or extracellular fibril assembly (Supplementary Fig. 2). As previously reported, Crtap−/− mice exhibit reduced bone mass and abnormal trabecular bone parameters (Fig. 2a,b)5. MicroCT imaging analysis of vertebrae and femurs demonstrated that compared with control Crtap−/− mice, TGFβ inhibition significantly improved trabecular bone parameters to near WT levels (Fig. 2a,b and Supplementary Table 1,2). The effects of TGFβ inhibition on the skeleton with 1D11 have been reported previously in WT and E-selectin ligand-1 knockout (Esl-1−/−) mice9, a model with higher TGFβ activity due to a defect in TGFβ production. While 1D11 moderately elevated bone volume/tissue volume (BV/TV) by 33% in WT mice, Esl-1−/− mice exhibited a 106% improvement. This suggests that targeting TGFβ in a pathophysiological situation where it is upregulated, could lead to a relatively more pronounced positive effect. In the present study, 1D11 improved the BV/TV at the spine by 235% in Crtap−/− mice, supporting that the dysregulated TGFβ signaling is a major contributor to the low bone mass.


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)

Phenotypic correction of Crtap−/− mice after treatment with the TGFβ neutralizing antibody 1D11. (a) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks (scale bar=500μm). (b) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=8 per group. (c) Histomorphometric analysis of L4 for osteoclast (N.Oc/BS) and osteoblast (N.Ob/BS) numbers per bone surface, and numbers of osteocytes per bone area (N.Ot/B.Ar) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=6 per group. (d) Hematoxylin/eosin staining of inflated lungs of 16-week-old wildtype (WT), control Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks. Representative images of n=8 mice per group are shown (scale bar=100 μm). (e) Quantification of the distance between alveolar structures by the mean-linear-intercept (MLI) method in lungs of WT, control Crtap−/− and 1D11-treated Crtap−/− mice. Results are shown as means±SDs, n=8 mice per group, 10 images analyzed per mouse. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.
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Figure 2: Phenotypic correction of Crtap−/− mice after treatment with the TGFβ neutralizing antibody 1D11. (a) MicroCT images of L4 vertebral bodies of 16-week-old wildtype (WT), control antibody-treated Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks (scale bar=500μm). (b) MicroCT analysis results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=8 per group. (c) Histomorphometric analysis of L4 for osteoclast (N.Oc/BS) and osteoblast (N.Ob/BS) numbers per bone surface, and numbers of osteocytes per bone area (N.Ot/B.Ar) in WT, control Crtap−/− and 1D11 treated Crtap−/− mice. Results are shown as means±SDs, n=6 per group. (d) Hematoxylin/eosin staining of inflated lungs of 16-week-old wildtype (WT), control Crtap−/−, and 1D11-treated Crtap−/− mice after treatment for 8 weeks. Representative images of n=8 mice per group are shown (scale bar=100 μm). (e) Quantification of the distance between alveolar structures by the mean-linear-intercept (MLI) method in lungs of WT, control Crtap−/− and 1D11-treated Crtap−/− mice. Results are shown as means±SDs, n=8 mice per group, 10 images analyzed per mouse. *P<0.05 for Crtap−/− vs. WT, #P<0.05 for Crtap−/− 1D11 vs. Crtap−/− control. NS, not significant.
Mentions: To understand whether upregulated TGFβ signaling represents a causal mechanism contributing to the bone and lung phenotypes in Crtap−/− mice, we performed a rescue experiment with a pan-TGFβ neutralizing antibody (1D11). Eight week old Crtap−/− mice were treated with 1D11 for eight weeks; control Crtap−/− and WT mice received a non-specific control antibody (13C4). 1D11 did not change body weight of the treated Crtap−/− mice, indicating that TGFβ inhibition did not affect their general nutritional status (Supplementary Fig. 1). Mass spectrometric and collagen cross-links analyses showed that 1D11 did not considerably change the status of P986 3-hydroxylation or collagen crosslinks in Crtap−/− mice, suggesting that dysregulated TGFβ signaling is a consequence of the altered collagen, and not directly involved in intracellular collagen processing or extracellular fibril assembly (Supplementary Fig. 2). As previously reported, Crtap−/− mice exhibit reduced bone mass and abnormal trabecular bone parameters (Fig. 2a,b)5. MicroCT imaging analysis of vertebrae and femurs demonstrated that compared with control Crtap−/− mice, TGFβ inhibition significantly improved trabecular bone parameters to near WT levels (Fig. 2a,b and Supplementary Table 1,2). The effects of TGFβ inhibition on the skeleton with 1D11 have been reported previously in WT and E-selectin ligand-1 knockout (Esl-1−/−) mice9, a model with higher TGFβ activity due to a defect in TGFβ production. While 1D11 moderately elevated bone volume/tissue volume (BV/TV) by 33% in WT mice, Esl-1−/− mice exhibited a 106% improvement. This suggests that targeting TGFβ in a pathophysiological situation where it is upregulated, could lead to a relatively more pronounced positive effect. In the present study, 1D11 improved the BV/TV at the spine by 235% in Crtap−/− mice, supporting that the dysregulated TGFβ signaling is a major contributor to the low bone mass.

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