Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1

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.

Figure 1. Uncontrolled Ank, Enpp1, Opn expression and increased pyrophosphate production in Nf1–deficient osteoblasts

(a) Nf1 mRNA expression in BMSCs differentiated for 7, 14 and 21 days (n = 3). (b) Extracellular PPi concentration in the conditioned medium of undifferentiated BMSCs (n = 3). (c) Ank, Enpp1 and Opn mRNA expression in BMSCs treated with vehicle (DMSO) or U0126 for 24 h (n = 3). (d). Ank, Enpp1 and Opn mRNA expression in long bones, calvariae and epiphyses of 3 week–old WT (blue bars) and Col2-Nf1 KO mice (grey bars)(n = 6). (e, f) ENPP1 and 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 from Col2-Nf1 KO mice, *:p < 0.05. ns: non–significant.

Figure 2. Altered pyrophosphate homeostasis in Nf1–deficient chondrocytes

(a) Hyperosteoidosis (pink, white arrow) in the primary spongiosa from Col2-Nf1 KO^−/− mice (undecalcified sections stained by von Kossa/Van Gieson, bar: 150 μm). (b) High–density chondrocyte pellets prepared from WT and Col2-Nf1 KO pups. Proteoglycan production (top panels, Alcian blue staining) and matrix mineralization (bottom panels, von Kossa staining)(n = 3. bar: 100 μm). (c) Ank, Enpp1 and Opn mRNA expression in high–density chondrocyte pellets (n = 3). (d) Relative extracellular PPi concentration, (e) ENPP1 activity and (f) ALP activity in WT and Col2-Nf1 KO high–density chondrocytes pellets (n = 3). *:p < 0.05.

Figure 3. Blunted BMP2 response and osteoblast differentiation potential in Nf1–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 and Ocn mRNA expression in BMSCs differentiated for 7, 14 and 21 days (n = 4). (c) Runx2 and Alpl mRNA expression in serum–starved BMSCs treated with vehicle (DMSO) or U0126 for 24 h (n = 6). (d) Extracellular PPi concentration/protein concentration in BMSCs differentiated for 7, 14 and 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.

Figure 4. BMP2 does not promote differentiation in Nf1–deficient BMSCs but 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 treatment for 1 h. Smad1/5 and β–actin served as loading control. (c and d) Alpl, Runx2, Col1a1, Ank, Enpp1 and Opn 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 BMP2 for 24 h (n = 3). (f and g) BMSC differentiation 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 2 weeks. 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.

Figure 5. sALP–FcD10 improves bone growth and cortical bone parameters in growing Col2-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: 250 μm) 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/Tissue Volume (hBV/TV, histology) in Col2-Nf1 KO newborn pups treated daily by sALP–FcD10 for 18 days (n > 8 mice/group). *:p < 0.05 versus WT; #:p < 0.05 versus vehicle in the same genotype group.

Figure 6. sALP–FcD10 improves trabecular bone mass, mineralization and bone structure in Osx-Nf1 KO mice

(a) Size of two month–old WT and Osx-Nf1 KO mice following doxycycline (Doxy) treatment from conception to P14. (b) Femoral hyperosteoidosis (pink stain following von Kossa/van Gieson staining), Osteoid Volume/Bone Volume ratio (OV/BV), Osteoid Surface/Bone Surface ratio (OS/BS) and Osteoid Thickness (O. Th) in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10 administration for 6 weeks (histomorphometric analyses, bar: 150 μm). (c) Femoral Bone Volume/Tissue Volume (BV/TV) in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10 administration (μCT). (d) Cortical porosity in Osx-Nf1 KO mice and partial beneficial effect of sALP–FcD10 administration (μCT). (e) Femoral cortical thickness in WT and Osx-Nf1 KO mice (μCT). (f) Moment of inertia in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10 administration (μCT). (g) Cortical Tissue Mineral Density (TMD) in WT and Osx-Nf1 KO mice (μCT). (h) Mineral–to–Collagen ratio (ν1 phosphate/Proline) in WT and Osx-Nf1 KO mice and rescue by sALP–FcD10 administration (Raman spectroscopy). (n > 8 mice/group). *:p < 0.05 versus WT; #:p < 0.05 versus vehicle in the same genotype group.