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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 bone growth and cortical bone parameters in growingCol2-Nf1 KO mice(a) BMSC matrix mineralization (CFU–Ob) and number (CFU–F)analyzed by Alizarin red–S and crystal violet staining, respectively (n= 3) following vehicle or sALP–FcD10 treatment for 2 weeks.(b–f) Bone growth (b, naso–anal length),vertebral (c, bar: 250 μm) and tibial (d, bar: 250μm) bone mineral density (X–rays), cortical thickness (e,Ct.Th, μCT), epiphyseal diameter (f, white arrow, bar: 45 μm,μCT) and hypertrophic zone von Kossa–positive calcified Bone Volume/TissueVolume (hBV/TV, histology) in Col2-Nf1 KO newborn pups treated daily bysALP–FcD10 for 18 days (n > 8 mice/group).*:p < 0.05 versus WT; #:p < 0.05versus vehicle in the same genotype group.
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Figure 5: sALP–FcD10 improves bone growth and cortical bone parameters in growingCol2-Nf1 KO mice(a) BMSC matrix mineralization (CFU–Ob) and number (CFU–F)analyzed by Alizarin red–S and crystal violet staining, respectively (n= 3) following vehicle or sALP–FcD10 treatment for 2 weeks.(b–f) Bone growth (b, naso–anal length),vertebral (c, bar: 250 μm) and tibial (d, bar: 250μm) bone mineral density (X–rays), cortical thickness (e,Ct.Th, μCT), epiphyseal diameter (f, white arrow, bar: 45 μm,μCT) and hypertrophic zone von Kossa–positive calcified Bone Volume/TissueVolume (hBV/TV, histology) in Col2-Nf1 KO newborn pups treated daily bysALP–FcD10 for 18 days (n > 8 mice/group).*:p < 0.05 versus WT; #:p < 0.05versus vehicle in the same genotype group.

Mentions: If excessive extracellular PPi levels cause the mineralization deficit observedin Col2-Nf1 KO mice, then reducing PPi concentration should havebeneficial effects on matrix mineralization. This is experimentally possible by inhibitingPPi generation or increasing its catabolism. The latter approach was chosen because PPi isa substrate for ALP and a recombinant form of human ALP (sALP–FcD10 orAsfotase–α) is clinically available to treatALPL–deficient subjects with hypophosphatasia42,43. We thustreated WT and Nf1–deficient BMSCs with vehicle orsALP–FcD10 (0.5 mg.ml−1) in osteogenic condition for 14 daysand assessed matrix mineralization. As predicted, sALP–FcD10 increased matrixmineralization in both genotypes, although the relative increase was more pronounced incultures from Col2-Nf1 KO versus WT mice (Fig. 5a), and despite the persistent differentiation deficit ofNf1–deficient BMSCs in the presence of sALP–FcD10(Supplementary Fig. 3a). Thistreatment reduced Opn expression inNf1–deficient BMSCs (Supplementary Fig. 3a), in agreement with theknown stimulatory effect of PPi on Opn expression25.


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 bone growth and cortical bone parameters in growingCol2-Nf1 KO mice(a) BMSC matrix mineralization (CFU–Ob) and number (CFU–F)analyzed by Alizarin red–S and crystal violet staining, respectively (n= 3) following vehicle or sALP–FcD10 treatment for 2 weeks.(b–f) Bone growth (b, naso–anal length),vertebral (c, bar: 250 μm) and tibial (d, bar: 250μm) bone mineral density (X–rays), cortical thickness (e,Ct.Th, μCT), epiphyseal diameter (f, white arrow, bar: 45 μm,μCT) and hypertrophic zone von Kossa–positive calcified Bone Volume/TissueVolume (hBV/TV, histology) in Col2-Nf1 KO newborn pups treated daily bysALP–FcD10 for 18 days (n > 8 mice/group).*:p < 0.05 versus WT; #:p < 0.05versus vehicle in the same genotype group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126855&req=5

Figure 5: sALP–FcD10 improves bone growth and cortical bone parameters in growingCol2-Nf1 KO mice(a) BMSC matrix mineralization (CFU–Ob) and number (CFU–F)analyzed by Alizarin red–S and crystal violet staining, respectively (n= 3) following vehicle or sALP–FcD10 treatment for 2 weeks.(b–f) Bone growth (b, naso–anal length),vertebral (c, bar: 250 μm) and tibial (d, bar: 250μm) bone mineral density (X–rays), cortical thickness (e,Ct.Th, μCT), epiphyseal diameter (f, white arrow, bar: 45 μm,μCT) and hypertrophic zone von Kossa–positive calcified Bone Volume/TissueVolume (hBV/TV, histology) in Col2-Nf1 KO newborn pups treated daily bysALP–FcD10 for 18 days (n > 8 mice/group).*:p < 0.05 versus WT; #:p < 0.05versus vehicle in the same genotype group.
Mentions: If excessive extracellular PPi levels cause the mineralization deficit observedin Col2-Nf1 KO mice, then reducing PPi concentration should havebeneficial effects on matrix mineralization. This is experimentally possible by inhibitingPPi generation or increasing its catabolism. The latter approach was chosen because PPi isa substrate for ALP and a recombinant form of human ALP (sALP–FcD10 orAsfotase–α) is clinically available to treatALPL–deficient subjects with hypophosphatasia42,43. We thustreated WT and Nf1–deficient BMSCs with vehicle orsALP–FcD10 (0.5 mg.ml−1) in osteogenic condition for 14 daysand assessed matrix mineralization. As predicted, sALP–FcD10 increased matrixmineralization in both genotypes, although the relative increase was more pronounced incultures from Col2-Nf1 KO versus WT mice (Fig. 5a), and despite the persistent differentiation deficit ofNf1–deficient BMSCs in the presence of sALP–FcD10(Supplementary Fig. 3a). Thistreatment reduced Opn expression inNf1–deficient BMSCs (Supplementary Fig. 3a), in agreement with theknown stimulatory effect of PPi on Opn expression25.

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