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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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Blunted BMP2 response and osteoblast differentiation potential inNf1–deficient osteoprogenitors(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob), crystal violet staining (cell number,CFU–F, left panel), soluble Alizarin redS/crystal violet optical density ratio(middle panel) and ALP activity/crystal violet ratio (right panel) (n= 6). (b) Runx2, Alpl andOcn mRNA expression in BMSCs differentiated for 7, 14 and 21 days(n = 4). (c) Runx2 andAlpl mRNA expression in serum–starved BMSCs treated withvehicle (DMSO) or U0126 for 24 h (n = 6). (d)Extracellular PPi concentration/protein concentration in BMSCs differentiated for 7, 14and 21 days (n = 4). (e) Normalized Ank,Enpp1 and Opn mRNA expression in BMSCs differentiated for 7,14 and 21 days (n = 4). Blue bars: WT mice, grey bars:Col2-Nf1 KO mice, *:p < 0.05.
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Figure 3: Blunted BMP2 response and osteoblast differentiation potential inNf1–deficient osteoprogenitors(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob), crystal violet staining (cell number,CFU–F, left panel), soluble Alizarin redS/crystal violet optical density ratio(middle panel) and ALP activity/crystal violet ratio (right panel) (n= 6). (b) Runx2, Alpl andOcn mRNA expression in BMSCs differentiated for 7, 14 and 21 days(n = 4). (c) Runx2 andAlpl mRNA expression in serum–starved BMSCs treated withvehicle (DMSO) or U0126 for 24 h (n = 6). (d)Extracellular PPi concentration/protein concentration in BMSCs differentiated for 7, 14and 21 days (n = 4). (e) Normalized Ank,Enpp1 and Opn mRNA expression in BMSCs differentiated for 7,14 and 21 days (n = 4). Blue bars: WT mice, grey bars:Col2-Nf1 KO mice, *:p < 0.05.

Mentions: BMSCs isolated from Col2-Nf1 KO mice displayed, compared toBMSCs isolated from WT mice, a significantly lower differentiation potential, as measuredby lower CFU–Ob colony number, TNSAP activity (Fig.3a) and lower expression of osteoblast differentiation markers includingRunx2, Alpl and Ocn (Fig. 3b). Similar results were obtained usingNf1flox/flox BMSCs infected with a cre–adenovirus(Supplementary Figs. 1d and e).In contrast to what was observed in the case of Ank andEnpp1 expression, however, MEK inhibition by U0126 (1 μM),Tremetinib or PD198306 (0.1 μM and 200 nM, respectively, data not shown) for 24 hdid not correct the expression level of Runx2 or Alpl inNf1–deficient BMSCs (Figs.3c), indicating that the expression of these two genes is not directly controlledby neurofibromin. Extracellular PPi concentration, as well as Ank,Enpp1 and Opn expression, remained above or equal toWT controls throughout the differentiation period (Fig. 3dand e).


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)

Blunted BMP2 response and osteoblast differentiation potential inNf1–deficient osteoprogenitors(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob), crystal violet staining (cell number,CFU–F, left panel), soluble Alizarin redS/crystal violet optical density ratio(middle panel) and ALP activity/crystal violet ratio (right panel) (n= 6). (b) Runx2, Alpl andOcn mRNA expression in BMSCs differentiated for 7, 14 and 21 days(n = 4). (c) Runx2 andAlpl mRNA expression in serum–starved BMSCs treated withvehicle (DMSO) or U0126 for 24 h (n = 6). (d)Extracellular PPi concentration/protein concentration in BMSCs differentiated for 7, 14and 21 days (n = 4). (e) Normalized Ank,Enpp1 and Opn mRNA expression in BMSCs differentiated for 7,14 and 21 days (n = 4). Blue bars: WT mice, grey bars:Col2-Nf1 KO mice, *:p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Blunted BMP2 response and osteoblast differentiation potential inNf1–deficient osteoprogenitors(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob), crystal violet staining (cell number,CFU–F, left panel), soluble Alizarin redS/crystal violet optical density ratio(middle panel) and ALP activity/crystal violet ratio (right panel) (n= 6). (b) Runx2, Alpl andOcn mRNA expression in BMSCs differentiated for 7, 14 and 21 days(n = 4). (c) Runx2 andAlpl mRNA expression in serum–starved BMSCs treated withvehicle (DMSO) or U0126 for 24 h (n = 6). (d)Extracellular PPi concentration/protein concentration in BMSCs differentiated for 7, 14and 21 days (n = 4). (e) Normalized Ank,Enpp1 and Opn mRNA expression in BMSCs differentiated for 7,14 and 21 days (n = 4). Blue bars: WT mice, grey bars:Col2-Nf1 KO mice, *:p < 0.05.
Mentions: BMSCs isolated from Col2-Nf1 KO mice displayed, compared toBMSCs isolated from WT mice, a significantly lower differentiation potential, as measuredby lower CFU–Ob colony number, TNSAP activity (Fig.3a) and lower expression of osteoblast differentiation markers includingRunx2, Alpl and Ocn (Fig. 3b). Similar results were obtained usingNf1flox/flox BMSCs infected with a cre–adenovirus(Supplementary Figs. 1d and e).In contrast to what was observed in the case of Ank andEnpp1 expression, however, MEK inhibition by U0126 (1 μM),Tremetinib or PD198306 (0.1 μM and 200 nM, respectively, data not shown) for 24 hdid not correct the expression level of Runx2 or Alpl inNf1–deficient BMSCs (Figs.3c), indicating that the expression of these two genes is not directly controlledby neurofibromin. Extracellular PPi concentration, as well as Ank,Enpp1 and Opn expression, remained above or equal toWT controls throughout the differentiation period (Fig. 3dand e).

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