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Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1.

de la Croix Ndong J, Makowski AJ, Uppuganti S, Vignaux G, Ono K, Perrien DS, Joubert S, Baglio SR, Granchi D, Stevenson DA, Rios JJ, Nyman JS, Elefteriou F - Nat. Med. (2014)

Bottom Line: NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin.The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration.These results establish neurofibromin as an essential regulator of bone mineralization.

View Article: PubMed Central - PubMed

Affiliation: 1] Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA. [2] Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

ABSTRACT
Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

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sALP–FcD10 improves trabecular bone mass, mineralization and bone structurein Osx-Nf1 KO mice(a) Size of two month–old WT and Osx-Nf1 KO micefollowing doxycycline (Doxy) treatment from conception to P14. (b) Femoralhyperosteoidosis (pink stain following von Kossa/van Gieson staining), Osteoid Volume/BoneVolume ratio (OV/BV), Osteoid Surface/Bone Surface ratio (OS/BS) and Osteoid Thickness (O.Th) in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10administration for 6 weeks (histomorphometric analyses, bar: 150 μm).(c) Femoral Bone Volume/Tissue Volume (BV/TV) in WT andOsx-Nf1 KO mice and rescue by sALP–FcD10 administration(μCT). (d) Cortical porosity in Osx-Nf1 KO mice andpartial beneficial effect of sALP–FcD10 administration (μCT).(e) Femoral cortical thickness in WT and Osx-Nf1 KO mice(μCT). (f) Moment of inertia in WT and Osx-Nf1 KOmice and rescue by sALP–FcD10 administration (μCT). (g)Cortical Tissue Mineral Density (TMD) in WT and Osx-Nf1 KO mice(μCT). (h) Mineral–to–Collagen ratio (ν1phosphate/Proline) in WT and Osx-Nf1 KO mice and rescue bysALP–FcD10 administration (Raman spectroscopy). (n > 8mice/group). *:p < 0.05 versus WT; #:p< 0.05 versus vehicle in the same genotype group.
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Figure 6: sALP–FcD10 improves trabecular bone mass, mineralization and bone structurein Osx-Nf1 KO mice(a) Size of two month–old WT and Osx-Nf1 KO micefollowing doxycycline (Doxy) treatment from conception to P14. (b) Femoralhyperosteoidosis (pink stain following von Kossa/van Gieson staining), Osteoid Volume/BoneVolume ratio (OV/BV), Osteoid Surface/Bone Surface ratio (OS/BS) and Osteoid Thickness (O.Th) in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10administration for 6 weeks (histomorphometric analyses, bar: 150 μm).(c) Femoral Bone Volume/Tissue Volume (BV/TV) in WT andOsx-Nf1 KO mice and rescue by sALP–FcD10 administration(μCT). (d) Cortical porosity in Osx-Nf1 KO mice andpartial beneficial effect of sALP–FcD10 administration (μCT).(e) Femoral cortical thickness in WT and Osx-Nf1 KO mice(μCT). (f) Moment of inertia in WT and Osx-Nf1 KOmice and rescue by sALP–FcD10 administration (μCT). (g)Cortical Tissue Mineral Density (TMD) in WT and Osx-Nf1 KO mice(μCT). (h) Mineral–to–Collagen ratio (ν1phosphate/Proline) in WT and Osx-Nf1 KO mice and rescue bysALP–FcD10 administration (Raman spectroscopy). (n > 8mice/group). *:p < 0.05 versus WT; #:p< 0.05 versus vehicle in the same genotype group.

Mentions: Because Col2-Nf1 KO mice manifest severe developmentalphenotypes that limit their survival, and thus the duration of treatments, we generatedmice in which Nf1 can be ablated postnatally in osteoprogenitors, usingthe inducible Tet-off–based Osx–cre transgenicmice46 crossed toNf1f/f mice47. This new mouse model makes it possible to dissect the mechanisms bywhich postnatal Nf1 ablation impairs bone homeostasis, withoutcomplications arising from developmental phenotypes.Osx–Nf1Osxf/f mice had a sizeundistinguishable from WT littermates upon doxycycline administration (i.e.cre–recombinase repression) from conception to day 14 (Fig. 6a) and had normal phosphate, calcium and 25OH vitamin Dserum concentrations (SupplementaryTable 1). Osx–cre–mediated Nf1ablation in osteoprogenitors at post–natal day 14 following doxycyclinewithdrawal, as seen in Col2-Nf1 KO mice, caused hyperosteoidosis (Fig. 6b), low bone mass (Fig. 6c), higher femoral diaphyseal cortical porosity (Fig. 6d), lower cortical thickness, mid–shaft moment ofinertia and cortical TMD (Fig. 6e–g).Cortical mineral–to–collagen ratio measured by Raman spectroscopy (Fig. 6h) was also lower in Osx-Nf1 KOmice, and femurs from Osx-Nf1 KO mice were mechanically weaker than thosefrom WT controls, as measured by a 3–point bending tests (Supplementary Table 2).


Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1.

de la Croix Ndong J, Makowski AJ, Uppuganti S, Vignaux G, Ono K, Perrien DS, Joubert S, Baglio SR, Granchi D, Stevenson DA, Rios JJ, Nyman JS, Elefteriou F - Nat. Med. (2014)

sALP–FcD10 improves trabecular bone mass, mineralization and bone structurein Osx-Nf1 KO mice(a) Size of two month–old WT and Osx-Nf1 KO micefollowing doxycycline (Doxy) treatment from conception to P14. (b) Femoralhyperosteoidosis (pink stain following von Kossa/van Gieson staining), Osteoid Volume/BoneVolume ratio (OV/BV), Osteoid Surface/Bone Surface ratio (OS/BS) and Osteoid Thickness (O.Th) in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10administration for 6 weeks (histomorphometric analyses, bar: 150 μm).(c) Femoral Bone Volume/Tissue Volume (BV/TV) in WT andOsx-Nf1 KO mice and rescue by sALP–FcD10 administration(μCT). (d) Cortical porosity in Osx-Nf1 KO mice andpartial beneficial effect of sALP–FcD10 administration (μCT).(e) Femoral cortical thickness in WT and Osx-Nf1 KO mice(μCT). (f) Moment of inertia in WT and Osx-Nf1 KOmice and rescue by sALP–FcD10 administration (μCT). (g)Cortical Tissue Mineral Density (TMD) in WT and Osx-Nf1 KO mice(μCT). (h) Mineral–to–Collagen ratio (ν1phosphate/Proline) in WT and Osx-Nf1 KO mice and rescue bysALP–FcD10 administration (Raman spectroscopy). (n > 8mice/group). *:p < 0.05 versus WT; #:p< 0.05 versus vehicle in the same genotype group.
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: sALP–FcD10 improves trabecular bone mass, mineralization and bone structurein Osx-Nf1 KO mice(a) Size of two month–old WT and Osx-Nf1 KO micefollowing doxycycline (Doxy) treatment from conception to P14. (b) Femoralhyperosteoidosis (pink stain following von Kossa/van Gieson staining), Osteoid Volume/BoneVolume ratio (OV/BV), Osteoid Surface/Bone Surface ratio (OS/BS) and Osteoid Thickness (O.Th) in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10administration for 6 weeks (histomorphometric analyses, bar: 150 μm).(c) Femoral Bone Volume/Tissue Volume (BV/TV) in WT andOsx-Nf1 KO mice and rescue by sALP–FcD10 administration(μCT). (d) Cortical porosity in Osx-Nf1 KO mice andpartial beneficial effect of sALP–FcD10 administration (μCT).(e) Femoral cortical thickness in WT and Osx-Nf1 KO mice(μCT). (f) Moment of inertia in WT and Osx-Nf1 KOmice and rescue by sALP–FcD10 administration (μCT). (g)Cortical Tissue Mineral Density (TMD) in WT and Osx-Nf1 KO mice(μCT). (h) Mineral–to–Collagen ratio (ν1phosphate/Proline) in WT and Osx-Nf1 KO mice and rescue bysALP–FcD10 administration (Raman spectroscopy). (n > 8mice/group). *:p < 0.05 versus WT; #:p< 0.05 versus vehicle in the same genotype group.
Mentions: Because Col2-Nf1 KO mice manifest severe developmentalphenotypes that limit their survival, and thus the duration of treatments, we generatedmice in which Nf1 can be ablated postnatally in osteoprogenitors, usingthe inducible Tet-off–based Osx–cre transgenicmice46 crossed toNf1f/f mice47. This new mouse model makes it possible to dissect the mechanisms bywhich postnatal Nf1 ablation impairs bone homeostasis, withoutcomplications arising from developmental phenotypes.Osx–Nf1Osxf/f mice had a sizeundistinguishable from WT littermates upon doxycycline administration (i.e.cre–recombinase repression) from conception to day 14 (Fig. 6a) and had normal phosphate, calcium and 25OH vitamin Dserum concentrations (SupplementaryTable 1). Osx–cre–mediated Nf1ablation in osteoprogenitors at post–natal day 14 following doxycyclinewithdrawal, as seen in Col2-Nf1 KO mice, caused hyperosteoidosis (Fig. 6b), low bone mass (Fig. 6c), higher femoral diaphyseal cortical porosity (Fig. 6d), lower cortical thickness, mid–shaft moment ofinertia and cortical TMD (Fig. 6e–g).Cortical mineral–to–collagen ratio measured by Raman spectroscopy (Fig. 6h) was also lower in Osx-Nf1 KOmice, and femurs from Osx-Nf1 KO mice were mechanically weaker than thosefrom WT controls, as measured by a 3–point bending tests (Supplementary Table 2).

Bottom Line: NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin.The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration.These results establish neurofibromin as an essential regulator of bone mineralization.

View Article: PubMed Central - PubMed

Affiliation: 1] Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA. [2] Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

ABSTRACT
Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Show MeSH
Related in: MedlinePlus