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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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BMP2 does not promote differentiation in Nf1–deficient BMSCsbut exacerbates their mineralization deficit(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob) and crystal violet (cell number,CFU–F) staining (n = 3) and ALP activity (n= 3), following vehicle or BMP2 treatment. (b)Phospho–Smad1/5 induction in serum–starved BMSCs following BMP2 treatmentfor 1 h. Smad1/5 and β–actin served as loading control. (cand d) Alpl, Runx2, Col1a1, Ank, Enpp1 andOpn mRNA expression following BMP2 treatment for 2 weeks (n= 3). (e) Extracellular PPi relative concentration(normalized to protein concentration) in the conditioned medium of BMSCs treated with BMP2for 24 h (n = 3). (f and g) BMSCdifferentiation analyzed by Alizarin red–S (differentiation/mineralization,CFU–Ob) and crystal violet (cell number, CFU–F) staining (f,n = 3) and ALP activity (g, n= 3) following treatment with vehicle or BMP2 or U0126 or both for 2weeks. Blue bars: WT mice; grey bars: Col2-Nf1 KO mice.*:p < 0.05 versus WT in the same treatment group;#:p < 0.05 versus vehicle in the same genotype group.
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Figure 4: BMP2 does not promote differentiation in Nf1–deficient BMSCsbut exacerbates their mineralization deficit(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob) and crystal violet (cell number,CFU–F) staining (n = 3) and ALP activity (n= 3), following vehicle or BMP2 treatment. (b)Phospho–Smad1/5 induction in serum–starved BMSCs following BMP2 treatmentfor 1 h. Smad1/5 and β–actin served as loading control. (cand d) Alpl, Runx2, Col1a1, Ank, Enpp1 andOpn mRNA expression following BMP2 treatment for 2 weeks (n= 3). (e) Extracellular PPi relative concentration(normalized to protein concentration) in the conditioned medium of BMSCs treated with BMP2for 24 h (n = 3). (f and g) BMSCdifferentiation analyzed by Alizarin red–S (differentiation/mineralization,CFU–Ob) and crystal violet (cell number, CFU–F) staining (f,n = 3) and ALP activity (g, n= 3) following treatment with vehicle or BMP2 or U0126 or both for 2weeks. Blue bars: WT mice; grey bars: Col2-Nf1 KO mice.*:p < 0.05 versus WT in the same treatment group;#:p < 0.05 versus vehicle in the same genotype group.

Mentions: Bone morphogenic proteins (BMPs) are known for their ability to promoteosteoprogenitor differentiation39 buthad limited effect on the differentiation of Nf1+/−osteoprogenitors and on bone union in Nf1+/−mice40,41. Recombinant hBMP2 (100 ng.ml−1) was unable tostimulate ALP activity, nor the formation of CFU–Ob in BMSC cultures fromCol2-Nf1 KO mice, although it did, as expected, promote CFU–Obformation and ALP activity in WT BMSC cultures, following 2 weeks of treatment (Fig. 4a). Smad1/5/8 phosphorylation in response to BMP2treatment (100 ng.ml−1, 1 h) was not affected by Nf1deficiency (Fig. 4b), indicating that the lack ofstimulatory effect of BMP2 on Nf1–deficient BMSC differentiationis not caused by repression of BMP2 receptor expression, or by the production of factor(s)inhibiting canonical signaling. Treatment with rhBMP2 for 2 weeks also failed to increasethe expression of Alpl, Runx2, and Col1a1 in BMSCcultures from Col2-Nf1 KO mice (Fig.4c). However, it significantly increased the expression of Ankand Enpp1 (but not Opn) (Fig. 4d) and PPi extracellular concentration (Fig.4e) in both WT and Nf1–deficient BMSCs. CFU–Obformation, ALP activity (Figs. 4f and g) and theexpression of Alpl and Col1a1 (Supplementary Figs. 2a and b) inNf1–deficient BMSC cultures were higher following a 2week–long combined treatment with the MEK inhibitor U0126 (1 μM) and BMP2(100 ng.ml−1), but not with either of these treatments alone. Thiscombination treatment also partially reduced the increased Ank, Enpp1 andPPi extracellular concentration detected in vehicle-treatedNf1–deficient BMSC cultures, possibly due to the antagonisticeffect of these two drugs on Ank and Enpp1 expression(Supplementary Figs 2c andd).


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)

BMP2 does not promote differentiation in Nf1–deficient BMSCsbut exacerbates their mineralization deficit(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob) and crystal violet (cell number,CFU–F) staining (n = 3) and ALP activity (n= 3), following vehicle or BMP2 treatment. (b)Phospho–Smad1/5 induction in serum–starved BMSCs following BMP2 treatmentfor 1 h. Smad1/5 and β–actin served as loading control. (cand d) Alpl, Runx2, Col1a1, Ank, Enpp1 andOpn mRNA expression following BMP2 treatment for 2 weeks (n= 3). (e) Extracellular PPi relative concentration(normalized to protein concentration) in the conditioned medium of BMSCs treated with BMP2for 24 h (n = 3). (f and g) BMSCdifferentiation analyzed by Alizarin red–S (differentiation/mineralization,CFU–Ob) and crystal violet (cell number, CFU–F) staining (f,n = 3) and ALP activity (g, n= 3) following treatment with vehicle or BMP2 or U0126 or both for 2weeks. Blue bars: WT mice; grey bars: Col2-Nf1 KO mice.*:p < 0.05 versus WT in the same treatment group;#:p < 0.05 versus vehicle in the same genotype group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126855&req=5

Figure 4: BMP2 does not promote differentiation in Nf1–deficient BMSCsbut exacerbates their mineralization deficit(a) BMSC differentiation analyzed by Alizarin red–S(differentiation/mineralization, CFU–Ob) and crystal violet (cell number,CFU–F) staining (n = 3) and ALP activity (n= 3), following vehicle or BMP2 treatment. (b)Phospho–Smad1/5 induction in serum–starved BMSCs following BMP2 treatmentfor 1 h. Smad1/5 and β–actin served as loading control. (cand d) Alpl, Runx2, Col1a1, Ank, Enpp1 andOpn mRNA expression following BMP2 treatment for 2 weeks (n= 3). (e) Extracellular PPi relative concentration(normalized to protein concentration) in the conditioned medium of BMSCs treated with BMP2for 24 h (n = 3). (f and g) BMSCdifferentiation analyzed by Alizarin red–S (differentiation/mineralization,CFU–Ob) and crystal violet (cell number, CFU–F) staining (f,n = 3) and ALP activity (g, n= 3) following treatment with vehicle or BMP2 or U0126 or both for 2weeks. Blue bars: WT mice; grey bars: Col2-Nf1 KO mice.*:p < 0.05 versus WT in the same treatment group;#:p < 0.05 versus vehicle in the same genotype group.
Mentions: Bone morphogenic proteins (BMPs) are known for their ability to promoteosteoprogenitor differentiation39 buthad limited effect on the differentiation of Nf1+/−osteoprogenitors and on bone union in Nf1+/−mice40,41. Recombinant hBMP2 (100 ng.ml−1) was unable tostimulate ALP activity, nor the formation of CFU–Ob in BMSC cultures fromCol2-Nf1 KO mice, although it did, as expected, promote CFU–Obformation and ALP activity in WT BMSC cultures, following 2 weeks of treatment (Fig. 4a). Smad1/5/8 phosphorylation in response to BMP2treatment (100 ng.ml−1, 1 h) was not affected by Nf1deficiency (Fig. 4b), indicating that the lack ofstimulatory effect of BMP2 on Nf1–deficient BMSC differentiationis not caused by repression of BMP2 receptor expression, or by the production of factor(s)inhibiting canonical signaling. Treatment with rhBMP2 for 2 weeks also failed to increasethe expression of Alpl, Runx2, and Col1a1 in BMSCcultures from Col2-Nf1 KO mice (Fig.4c). However, it significantly increased the expression of Ankand Enpp1 (but not Opn) (Fig. 4d) and PPi extracellular concentration (Fig.4e) in both WT and Nf1–deficient BMSCs. CFU–Obformation, ALP activity (Figs. 4f and g) and theexpression of Alpl and Col1a1 (Supplementary Figs. 2a and b) inNf1–deficient BMSC cultures were higher following a 2week–long combined treatment with the MEK inhibitor U0126 (1 μM) and BMP2(100 ng.ml−1), but not with either of these treatments alone. Thiscombination treatment also partially reduced the increased Ank, Enpp1 andPPi extracellular concentration detected in vehicle-treatedNf1–deficient BMSC cultures, possibly due to the antagonisticeffect of these two drugs on Ank and Enpp1 expression(Supplementary Figs 2c andd).

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