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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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Excessive TGFβ signaling in Crtap−/− mice. (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Crtap−/− mice. Results are shown as fold change of the mean of WT group±SD; n=5 per group. (b) Western blot analysis of P3 calvarial protein extracts showing amounts of activated Smad2 (pSmad2) relative to total Smad2 protein in Crtap−/− versus WT mice; n=3 per group. (c) Quantification of the Western blot shown in b. Results are shown as fold change of the mean of WT group±SD. (d) Bioluminescence in regions that overlap with skeletal structures in Crtap−/− compared with WT mice that were intercrossed to TGFβ-reporter mice (SBE-Luc mice). Representative image of 3 litters at P10 is shown (scale bar=1cm). (e) TGFβ activity in conditioned medium from WT and Crtap−/− bone marrow stromal cells cultured under osteogenic conditions. Results are shown as fold change of the mean of WT group±SD, n=6 per group. (f) Immunostaining of lungs (P10) for pSmad2 (red) in WT and Crtap−/− mice, DAPI (blue) staining of nuclei (40X magnification). Representative images of n=3 mice per group are shown (scale bar=20μm). *P<0.05 WT versus Crtap−/−.
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Figure 1: Excessive TGFβ signaling in Crtap−/− mice. (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Crtap−/− mice. Results are shown as fold change of the mean of WT group±SD; n=5 per group. (b) Western blot analysis of P3 calvarial protein extracts showing amounts of activated Smad2 (pSmad2) relative to total Smad2 protein in Crtap−/− versus WT mice; n=3 per group. (c) Quantification of the Western blot shown in b. Results are shown as fold change of the mean of WT group±SD. (d) Bioluminescence in regions that overlap with skeletal structures in Crtap−/− compared with WT mice that were intercrossed to TGFβ-reporter mice (SBE-Luc mice). Representative image of 3 litters at P10 is shown (scale bar=1cm). (e) TGFβ activity in conditioned medium from WT and Crtap−/− bone marrow stromal cells cultured under osteogenic conditions. Results are shown as fold change of the mean of WT group±SD, n=6 per group. (f) Immunostaining of lungs (P10) for pSmad2 (red) in WT and Crtap−/− mice, DAPI (blue) staining of nuclei (40X magnification). Representative images of n=3 mice per group are shown (scale bar=20μm). *P<0.05 WT versus Crtap−/−.

Mentions: Compared with wild type (WT) samples, calvarial bone of Crtap−/− mice showed a higher expression of the TGFβ targets Cdkn1a (cyclin-dependent kinase inhibitor 1a, P21) and Serpine1 (plasminogen activator inhibitor-1), consistent with elevated TGFβ activity (Fig. 1a). To confirm activation of the intracellular TGFβ signaling pathway, we evaluated the status of Smad2, a second messenger protein, which becomes phosphorylated after activation of TGFβ receptors. Consistently, immunoblot analyses demonstrated a greater ratio of phosphorylated Smad2 (pSmad2) to total Smad2 in calvarial bone samples of Crtap−/− compared with WT mice, indicating higher TGFβ signaling (Fig. 1b,c). To confirm higher TGFβ activity in vivo, we intercrossed Crtap−/− mice with reporter mice expressing luciferase in response to TGFβ (SBE-luc mice). Compared with WT/SBE-luc littermates, Crtap−/−/SBE-luc mice showed a more intense bioluminescence of areas over skeletal structures, indicating higher TGFβ activity in vivo (Fig. 1d; in 3 litters Crtap−/− mice show a mean 2.86 fold (SD±0.34) bioluminescence signal at the head/calvaria compared with WT mice). To test whether the higher TGFβ signaling is intrinsic to bone, i.e. tissue autonomous, we cultured bone marrow stromal cells (BMSCs) under osteogenic conditions in vitro. By using a TGFβ reporter cell line, we found that conditioned medium from Crtap−/− BMSCs exhibited greater TGFβ activity compared with medium from WT BMSCs (Fig. 1e). Together, these findings indicate that loss of Crtap enhances TGFβ signaling in bone in a tissue autonomous fashion.


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)

Excessive TGFβ signaling in Crtap−/− mice. (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Crtap−/− mice. Results are shown as fold change of the mean of WT group±SD; n=5 per group. (b) Western blot analysis of P3 calvarial protein extracts showing amounts of activated Smad2 (pSmad2) relative to total Smad2 protein in Crtap−/− versus WT mice; n=3 per group. (c) Quantification of the Western blot shown in b. Results are shown as fold change of the mean of WT group±SD. (d) Bioluminescence in regions that overlap with skeletal structures in Crtap−/− compared with WT mice that were intercrossed to TGFβ-reporter mice (SBE-Luc mice). Representative image of 3 litters at P10 is shown (scale bar=1cm). (e) TGFβ activity in conditioned medium from WT and Crtap−/− bone marrow stromal cells cultured under osteogenic conditions. Results are shown as fold change of the mean of WT group±SD, n=6 per group. (f) Immunostaining of lungs (P10) for pSmad2 (red) in WT and Crtap−/− mice, DAPI (blue) staining of nuclei (40X magnification). Representative images of n=3 mice per group are shown (scale bar=20μm). *P<0.05 WT versus Crtap−/−.
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Figure 1: Excessive TGFβ signaling in Crtap−/− mice. (a) Quantitative RT-PCR of TGFβ target genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Crtap−/− mice. Results are shown as fold change of the mean of WT group±SD; n=5 per group. (b) Western blot analysis of P3 calvarial protein extracts showing amounts of activated Smad2 (pSmad2) relative to total Smad2 protein in Crtap−/− versus WT mice; n=3 per group. (c) Quantification of the Western blot shown in b. Results are shown as fold change of the mean of WT group±SD. (d) Bioluminescence in regions that overlap with skeletal structures in Crtap−/− compared with WT mice that were intercrossed to TGFβ-reporter mice (SBE-Luc mice). Representative image of 3 litters at P10 is shown (scale bar=1cm). (e) TGFβ activity in conditioned medium from WT and Crtap−/− bone marrow stromal cells cultured under osteogenic conditions. Results are shown as fold change of the mean of WT group±SD, n=6 per group. (f) Immunostaining of lungs (P10) for pSmad2 (red) in WT and Crtap−/− mice, DAPI (blue) staining of nuclei (40X magnification). Representative images of n=3 mice per group are shown (scale bar=20μm). *P<0.05 WT versus Crtap−/−.
Mentions: Compared with wild type (WT) samples, calvarial bone of Crtap−/− mice showed a higher expression of the TGFβ targets Cdkn1a (cyclin-dependent kinase inhibitor 1a, P21) and Serpine1 (plasminogen activator inhibitor-1), consistent with elevated TGFβ activity (Fig. 1a). To confirm activation of the intracellular TGFβ signaling pathway, we evaluated the status of Smad2, a second messenger protein, which becomes phosphorylated after activation of TGFβ receptors. Consistently, immunoblot analyses demonstrated a greater ratio of phosphorylated Smad2 (pSmad2) to total Smad2 in calvarial bone samples of Crtap−/− compared with WT mice, indicating higher TGFβ signaling (Fig. 1b,c). To confirm higher TGFβ activity in vivo, we intercrossed Crtap−/− mice with reporter mice expressing luciferase in response to TGFβ (SBE-luc mice). Compared with WT/SBE-luc littermates, Crtap−/−/SBE-luc mice showed a more intense bioluminescence of areas over skeletal structures, indicating higher TGFβ activity in vivo (Fig. 1d; in 3 litters Crtap−/− mice show a mean 2.86 fold (SD±0.34) bioluminescence signal at the head/calvaria compared with WT mice). To test whether the higher TGFβ signaling is intrinsic to bone, i.e. tissue autonomous, we cultured bone marrow stromal cells (BMSCs) under osteogenic conditions in vitro. By using a TGFβ reporter cell line, we found that conditioned medium from Crtap−/− BMSCs exhibited greater TGFβ activity compared with medium from WT BMSCs (Fig. 1e). Together, these findings indicate that loss of Crtap enhances TGFβ signaling in bone in a tissue autonomous fashion.

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