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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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Altered pyrophosphate homeostasis in Nf1–deficientchondrocytes(a) Hyperosteoidosis (pink, white arrow) in the primary spongiosa fromCol2-Nf1 KO−/− mice (undecalcified sectionsstained by von Kossa/Van Gieson, bar: 150 μm). (b)High–density chondrocyte pellets prepared from WT and Col2-Nf1 KOpups. Proteoglycan production (top panels, Alcian blue staining) and matrix mineralization(bottom panels, von Kossa staining)(n = 3. bar: 100 μm).(c) Ank, Enpp1 and OpnmRNA expression in high–density chondrocyte pellets (n =3). (d) Relative extracellular PPi concentration, (e) ENPP1activity and (f) ALP activity in WT and Col2-Nf1 KOhigh–density chondrocytes pellets (n = 3).*:p < 0.05.
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Figure 2: Altered pyrophosphate homeostasis in Nf1–deficientchondrocytes(a) Hyperosteoidosis (pink, white arrow) in the primary spongiosa fromCol2-Nf1 KO−/− mice (undecalcified sectionsstained by von Kossa/Van Gieson, bar: 150 μm). (b)High–density chondrocyte pellets prepared from WT and Col2-Nf1 KOpups. Proteoglycan production (top panels, Alcian blue staining) and matrix mineralization(bottom panels, von Kossa staining)(n = 3. bar: 100 μm).(c) Ank, Enpp1 and OpnmRNA expression in high–density chondrocyte pellets (n =3). (d) Relative extracellular PPi concentration, (e) ENPP1activity and (f) ALP activity in WT and Col2-Nf1 KOhigh–density chondrocytes pellets (n = 3).*:p < 0.05.

Mentions: Mice lacking Nf1 in mature osteoblasts(Col1-Nf1 KO) have a uniform distribution ofnon–mineralized matrix throughout trabecular bone compartments18, whereas mice lacking Nf1 inosteochondroprogenitors and chondrocytes are characterized by an osteoid preferentiallydistributed in the primary spongiosa, where osteoblasts and chondrocytes mineralize theirmatrix (Fig. 2a). Based on these observations andbecause neurofibromin is expressed in hypertrophic chondrocytes37,38, wehypothesized that this RAS–GAP could also contribute to cartilage mineralization,which is a process important for bone growth and ossification during development and bonehealing in adults. In support of this hypothesis, Col2-Nf1 KO chondrocytehigh–density micromass cultures generated a typical Alcian blue–positivematrix but did not show signs of mineralization, in contrast to WT chondrocyte cultures(Fig. 2b). In addition, Ank, Enpp1and Opn expression was significantly higher inNf1–deficient micromass chondrocyte cultures versus WT cultures(Fig. 2c), in agreement with the data obtained fromcartilaginous epiphyses, which contain a high proportion of chondrocytes (Fig. 1d). Accordingly, extracellular PPi concentration (Fig. 2d) and Enpp1 enzymatic activity (Fig. 2e) were significantly higher, whereas ALP activity was lower(Fig. 2f) in Nf1–deficientversus WT chondrocytes.


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)

Altered pyrophosphate homeostasis in Nf1–deficientchondrocytes(a) Hyperosteoidosis (pink, white arrow) in the primary spongiosa fromCol2-Nf1 KO−/− mice (undecalcified sectionsstained by von Kossa/Van Gieson, bar: 150 μm). (b)High–density chondrocyte pellets prepared from WT and Col2-Nf1 KOpups. Proteoglycan production (top panels, Alcian blue staining) and matrix mineralization(bottom panels, von Kossa staining)(n = 3. bar: 100 μm).(c) Ank, Enpp1 and OpnmRNA expression in high–density chondrocyte pellets (n =3). (d) Relative extracellular PPi concentration, (e) ENPP1activity and (f) ALP activity in WT and Col2-Nf1 KOhigh–density chondrocytes pellets (n = 3).*:p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Altered pyrophosphate homeostasis in Nf1–deficientchondrocytes(a) Hyperosteoidosis (pink, white arrow) in the primary spongiosa fromCol2-Nf1 KO−/− mice (undecalcified sectionsstained by von Kossa/Van Gieson, bar: 150 μm). (b)High–density chondrocyte pellets prepared from WT and Col2-Nf1 KOpups. Proteoglycan production (top panels, Alcian blue staining) and matrix mineralization(bottom panels, von Kossa staining)(n = 3. bar: 100 μm).(c) Ank, Enpp1 and OpnmRNA expression in high–density chondrocyte pellets (n =3). (d) Relative extracellular PPi concentration, (e) ENPP1activity and (f) ALP activity in WT and Col2-Nf1 KOhigh–density chondrocytes pellets (n = 3).*:p < 0.05.
Mentions: Mice lacking Nf1 in mature osteoblasts(Col1-Nf1 KO) have a uniform distribution ofnon–mineralized matrix throughout trabecular bone compartments18, whereas mice lacking Nf1 inosteochondroprogenitors and chondrocytes are characterized by an osteoid preferentiallydistributed in the primary spongiosa, where osteoblasts and chondrocytes mineralize theirmatrix (Fig. 2a). Based on these observations andbecause neurofibromin is expressed in hypertrophic chondrocytes37,38, wehypothesized that this RAS–GAP could also contribute to cartilage mineralization,which is a process important for bone growth and ossification during development and bonehealing in adults. In support of this hypothesis, Col2-Nf1 KO chondrocytehigh–density micromass cultures generated a typical Alcian blue–positivematrix but did not show signs of mineralization, in contrast to WT chondrocyte cultures(Fig. 2b). In addition, Ank, Enpp1and Opn expression was significantly higher inNf1–deficient micromass chondrocyte cultures versus WT cultures(Fig. 2c), in agreement with the data obtained fromcartilaginous epiphyses, which contain a high proportion of chondrocytes (Fig. 1d). Accordingly, extracellular PPi concentration (Fig. 2d) and Enpp1 enzymatic activity (Fig. 2e) were significantly higher, whereas ALP activity was lower(Fig. 2f) in Nf1–deficientversus WT chondrocytes.

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