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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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Uncontrolled Ank, Enpp1, Opn expression and increased pyrophosphateproduction in Nf1–deficient osteoblasts(a) Nf1 mRNA expression in BMSCs differentiated for 7, 14and 21 days (n = 3). (b) Extracellular PPiconcentration in the conditioned medium of undifferentiated BMSCs (n= 3). (c) Ank, Enpp1 andOpn mRNA expression in BMSCs treated with vehicle (DMSO) or U0126 for24 h (n = 3). (d). Ank,Enpp1 and Opn mRNA expression in long bones, calvariaeand epiphyses of 3 week–old WT (blue bars) and Col2-Nf1 KO mice(grey bars)(n = 6). (e, f) ENPP1and ANK mRNA expression in bone marrow adherent cells from control(n = 6) and NF1 pseudarthrosis (PA, n= 9) biopsies. Blue bars: BMSCs from WT mice, grey bars: BMSCs fromCol2-Nf1 KO mice, *:p < 0.05. ns:non–significant.
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Figure 1: Uncontrolled Ank, Enpp1, Opn expression and increased pyrophosphateproduction in Nf1–deficient osteoblasts(a) Nf1 mRNA expression in BMSCs differentiated for 7, 14and 21 days (n = 3). (b) Extracellular PPiconcentration in the conditioned medium of undifferentiated BMSCs (n= 3). (c) Ank, Enpp1 andOpn mRNA expression in BMSCs treated with vehicle (DMSO) or U0126 for24 h (n = 3). (d). Ank,Enpp1 and Opn mRNA expression in long bones, calvariaeand epiphyses of 3 week–old WT (blue bars) and Col2-Nf1 KO mice(grey bars)(n = 6). (e, f) ENPP1and ANK mRNA expression in bone marrow adherent cells from control(n = 6) and NF1 pseudarthrosis (PA, n= 9) biopsies. Blue bars: BMSCs from WT mice, grey bars: BMSCs fromCol2-Nf1 KO mice, *:p < 0.05. ns:non–significant.

Mentions: To address if and how Nf1 regulates bone mineralization, wefirst asked whether Nf1 ablation in bone marrow stromal cells (BMSCs)affects extracellular PPi concentrations. BMSCs from Col2-Nf1 KO mice,lacking Nf1 in osteochondroprogenitor cells, were characterized by a60–70% lower Nf1 expression compared to WT mice (Fig. 1a), consistent with the heterogeneous nature ofthese cultures36. This lowerNf1 expression level was accompanied by a significant 70%higher extracellular PPI concentration in the conditioned medium (CM) of undifferentiatedBMSC cultures compared to WT controls (Fig. 1b).Addition of a recombinant form of ALP (sALP–FcD10, 0.5μg.ml−1, see below) to induce PPi hydrolysis significantlyreduced the amount of PPi detected in both genotypes, confirming the validity of the PPimeasurements.


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)

Uncontrolled Ank, Enpp1, Opn expression and increased pyrophosphateproduction in Nf1–deficient osteoblasts(a) Nf1 mRNA expression in BMSCs differentiated for 7, 14and 21 days (n = 3). (b) Extracellular PPiconcentration in the conditioned medium of undifferentiated BMSCs (n= 3). (c) Ank, Enpp1 andOpn mRNA expression in BMSCs treated with vehicle (DMSO) or U0126 for24 h (n = 3). (d). Ank,Enpp1 and Opn mRNA expression in long bones, calvariaeand epiphyses of 3 week–old WT (blue bars) and Col2-Nf1 KO mice(grey bars)(n = 6). (e, f) ENPP1and ANK mRNA expression in bone marrow adherent cells from control(n = 6) and NF1 pseudarthrosis (PA, n= 9) biopsies. Blue bars: BMSCs from WT mice, grey bars: BMSCs fromCol2-Nf1 KO mice, *:p < 0.05. ns:non–significant.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Uncontrolled Ank, Enpp1, Opn expression and increased pyrophosphateproduction in Nf1–deficient osteoblasts(a) Nf1 mRNA expression in BMSCs differentiated for 7, 14and 21 days (n = 3). (b) Extracellular PPiconcentration in the conditioned medium of undifferentiated BMSCs (n= 3). (c) Ank, Enpp1 andOpn mRNA expression in BMSCs treated with vehicle (DMSO) or U0126 for24 h (n = 3). (d). Ank,Enpp1 and Opn mRNA expression in long bones, calvariaeand epiphyses of 3 week–old WT (blue bars) and Col2-Nf1 KO mice(grey bars)(n = 6). (e, f) ENPP1and ANK mRNA expression in bone marrow adherent cells from control(n = 6) and NF1 pseudarthrosis (PA, n= 9) biopsies. Blue bars: BMSCs from WT mice, grey bars: BMSCs fromCol2-Nf1 KO mice, *:p < 0.05. ns:non–significant.
Mentions: To address if and how Nf1 regulates bone mineralization, wefirst asked whether Nf1 ablation in bone marrow stromal cells (BMSCs)affects extracellular PPi concentrations. BMSCs from Col2-Nf1 KO mice,lacking Nf1 in osteochondroprogenitor cells, were characterized by a60–70% lower Nf1 expression compared to WT mice (Fig. 1a), consistent with the heterogeneous nature ofthese cultures36. This lowerNf1 expression level was accompanied by a significant 70%higher extracellular PPI concentration in the conditioned medium (CM) of undifferentiatedBMSC cultures compared to WT controls (Fig. 1b).Addition of a recombinant form of ALP (sALP–FcD10, 0.5μg.ml−1, see below) to induce PPi hydrolysis significantlyreduced the amount of PPi detected in both genotypes, confirming the validity of the PPimeasurements.

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