Mice lacking Nf1 in osteochondroprogenitor cells display skeletal dysplasia similar to patients with neurofibromatosis type I - PubMed (original) (raw)

. 2011 Oct 15;20(20):3910-24.

doi: 10.1093/hmg/ddr310. Epub 2011 Jul 14.

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Mice lacking Nf1 in osteochondroprogenitor cells display skeletal dysplasia similar to patients with neurofibromatosis type I

Weixi Wang et al. Hum Mol Genet. 2011.

Abstract

Mutations in NF1 cause neurofibromatosis type I (NF1), a disorder characterized, among other clinical manifestations, by generalized and focal bony lesions. Dystrophic scoliosis and tibial pseudoarthrosis are the most severe skeletal manifestations for which treatment is not satisfactory, emphasizing the dearth of knowledge related to the biology of NF1 in bone cells. Using reporter mice, we report here that the mouse Col2α1-Cre promoter (collagen, type II, alpha 1) is active not only in chondrocytes but also in adult bone marrow osteoprogenitors giving rise to osteoblasts. Based on this finding, we crossed the Col2α1-Cre transgenic and Nf1(flox/flox) mice to determine whether loss of Nf1 in axial and appendicular osteochondroprogenitors recapitulates the skeletal abnormalities of NF1 patients. By microtomographic and X-rays studies, we show that Nf1(Col2)(-/-) mice display progressive scoliosis and kyphosis, tibial bowing and abnormalities in skull and anterior chest wall formation. These defects were accompanied by a low bone mass phenotype, high bone cortical porosity, osteoidosis, increased osteoclastogenesis and decreased osteoblast number, as quantified by histomorphometry and 3D-microtomography. Loss of Nf1 in osteochondroprogenitors also caused severe short stature and intervertebral disc defects. Blockade of the RAS/ERK activation characteristic of Nf1(-/-) osteoprogenitors by lovastatin during embryonic development could attenuate the increased cortical porosity observed in mutant pups. These data and the skeletal similarities between this mouse model and NF1 patients thus suggest that activation of the RAS/ERK pathway by Nf1 loss-of-function in osteochondroprogenitors is responsible for the vertebral and tibia lesions in NF1 patients, and that this molecular signature may represent a good therapeutic target.

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Figures

Figure 1.

Figure 1.

Neurofibromin is expressed in osteochondroprogenitor cells and their progeny. (A) Nf1 mRNA expression was detected by RT-PCR in multiple primary cells and cell lines including adherent bone marrow osteochondroprogenitors (BMSCs and C3H10T1/2), chondrocytes (Ch: rib primary chondrocytes and TMC23 cells) and osteoblasts (OB) (calvaria osteoblasts and MC3T3 cells). (B) Neurofibromin expression (brown staining) was detected in growth plate hypertrophic chondrocytes, trabecular osteoblasts and cells of the periosteum and perichondrium (immunohistochemistry).

Figure 2.

Figure 2.

The Col2α1Cre promoter is active in Nf1 flox/flox osteochondroprogenitor cells. (A) X-gal staining of BMSCs from Col2α1-Cre;R26R mice at day 1, 3, 6, 9 and 13. (B) X-gal and von Kossa co-staining of BMSC cultures from R26R and Col2α1-Cre;R26R mice following 21 days of osteogenic differentiation. (C) X-gal and nuclear red staining of 2-month-old R26R and Col2α1-Cre;R26R long bones. (D) Non-recombined (N-Rec.) and recombined (Rec.) Nf1 alleles detected by PCR using genomic DNA from bone tissues and primary cells. The ratio of Rec./N.Rec. was calculated following densitometry.

Figure 3.

Figure 3.

_Nf1Col2_−/− mice display severe short stature and defects of endochondral bone formation. (A) _Nf1Col2_−/− mice were born (P0) with a size similar to WT littermates. (B) Increasing growth retardation in _Nf1Col2_−/− mice over a 2-month-long period. (C) _Nf1Col2_−/− mice were half the size of WT littermates by 1 month of age. (D) Shortening of humeri, radii, ulnae (top left), scapulae (bottom left), tibiae (top right) and femurs (bottom right) in 1-month-old _Nf1Col2_−/− mice. (E) Reduced size of the hypertrophic zone in the growth plate of P0 _Nf1Col2_−/− mice compared with WT littermates, assessed by in situ hybridization for the indicated genes. (F) Anterolateral tibia bowing in _Nf1Col2_−/− mice. (G and H) Anterior chest wall anomaly in 2-month-old _Nf1Col2_−/− mice. Arrows point to the protruding and fused anterior bone element. (I) Defect of skull structure in 2-month-old WT and _Nf1Col2_−/− mice (3D-microtomographic images). *P < 0.01 versus WT mice, n = 5.

Figure 4.

Figure 4.

Lack of Nf1 causes IVD formation defects. (A) 90° rotation of the NP in the vertebral axis of 1-month-old _Nf1Col2_−/− mice (hematoxylin and eosin staining). (B) Discrete patterning of the spinal column in E13.5 _Nf1Col2_−/− embryos (skeletal preparations). Arrows point to the forming IVD. (C) Unsegmented notochord in E14.5 _Nf1Col2_−/− embryos (Alcian blue and Fast Red staining). (D) Decreased apoptotic cell number (arrow) in the notochords of E14.5 _Nf1Col2_−/− embryos (TUNEL assay). (E) Decreased proliferation in the NP and sclerotome area of _Nf1Col2_−/− embryos (BrdU labeling). np, nucleus pulposus; sc, sclerotome. (F) Reduced size and delayed regression of the nucleus pulposus (arrow) in E16.5 _Nf1Col2_−/− embryos (skeletal preparations). (G) Abnormal IVD structure (arrow) in _Nf1Col2_−/− mice from E16.5 to P0 (blue and Fast Red staining). *P < 0.01 versus WT mice, n = 5.

Figure 5.

Figure 5.

Neurofibromin is required for proper axial skeleton formation. (A) Absence of ossification centers (arrow) in E16.5 _Nf1Col2_−/− embryonic VB (skeletal preparations). (B) _Nf1Col2_−/− mice displayed a 45° caudal angulation (arrow) at 6 months of age (X-rays). (C) Vertebral fusion at the base of the tail (arrow) in 1-month-old _Nf1Col2_−/− mice (X-rays). (D) Misalignments between vertebral bodies by 1 month of age (arrow), scoliosis by 3 months of age (arrow) and vertebral fusion and severe loss of BMD (arrow) by 6 months of age in _Nf1Col2_−/− mice (X-rays). (E) Kyphosis was observed in _Nf1Col2_−/− mice by 3 months of age (X-rays). (F) Abnormal VB shape in _Nf1Col2_−/− mice (3D-microtomography) at 6 months of age.

Figure 6.

Figure 6.

Lack of Nf1 in osteochondroprogenitors impairs bone structure and remodeling. Cortical porosity in the skull (A), long bones (B) and vertebrae (C) of 3-week-old _Nf1Col2_−/− mice (3D-microtomography). (D) Rankl and Opg expression in 2-month-old WT and _Nf1Col2_−/− lumbar vertebrae measured by qPCR, n = 4. (E) Osteoidosis in 2-month-old _Nf1Col2_−/− femurs. (F) Decreased femoral trabecular BV/TV, thickness and number and (G) decreased cortical mineral BMD (mBMD), thickness (Cor Th) and MOI in 2-month-old _Nf1Col2_−/− mice (3D-microtomography). *P < 0.01 versus WT mice, n = 7.

Figure 7.

Figure 7.

High cortical porosity in an NF1 patient presenting with pseudoarthrosis. Images are of the proximal portion of a surgically discarded tibial sample of a 3-year-old child with NF1 who had a fracture of the tibia with subsequent non-union and pseudoarthrosis. Control sample is from a dissected portion of the tibia near the junction of the middle and distal third segment in a 6-year-old deceased child without NF1 or chronic medical conditions. Fixed bone samples were staged in preparation for scanning and placed in a bench-top microCT scanner (Scanco µCT40, Zurich, Switzerland). The samples were scanned at optimized parameters (including resolution, power and frame averages) to derive the images and metrics for the anatomical region of interest. Three-dimensional volume rendering snapshots (left panel) and 2D cross-section snapshots (right panel) of the tibial samples (A: control individual, B: NF1 individual). Procedures for human sample collection and analyses were approved by the University of Utah Institutional Review Board.

Figure 8.

Figure 8.

Lovastatin treatment attenuates the cortical porosity in _Nf1Col2_−/− mice. (A) Activated Erk1/2 phosphorylation in _Nf1_−/− BMSCs was corrected by lovastatin treatment. (B) Lovastatin in utero treatment from E10.5 to P0 significantly decreased cortical porosity in P0 _Nf1Col2_−/− mice. (C) Increased cortical porosity and OV in P30 _Nf1Col2_−/− mice, and trend toward a beneficial effect of lovastatin administered in utero in 3-week-old _Nf1Col2_−/− mice. *P < 0.01 versus WT mice; #P < 0.01 versus _Nf1Col2_−/− mice, n = 7.

References

    1. Friedman J.M. Epidemiology of neurofibromatosis type 1. Am. J. Med. Genet. 1999;89:1–6. doi:10.1002/(SICI)1096-8628(19990326)89:1<1::AID-AJMG3>3.0.CO;2-8. - DOI - PubMed
    1. Upadhyaya M., Roberts S.H., Maynard J., Sorour E., Thompson P.W., Vaughan M., Wilkie A.O., Hughes H.E. A cytogenetic deletion, del(17)(q11.22q21.1), in a patient with sporadic neurofibromatosis type 1 (NF1) associated with dysmorphism and developmental delay. J. Med. Genet. 1996;33:148–152. doi:10.1136/jmg.33.2.148. - DOI - PMC - PubMed
    1. Xu G.F., O'Connell P., Viskochil D., Cawthon R., Robertson M., Culver M., Dunn D., Stevens J., Gesteland R., White R., et al. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990;62:599–608. doi:10.1016/0092-8674(90)90024-9. - DOI - PubMed
    1. Daston M.M., Scrable H., Nordlund M., Sturbaum A.K., Nissen L.M., Ratner N. The protein product of the neurofibromatosis type 1 gene is expressed at highest abundance in neurons, Schwann cells, and oligodendrocytes. Neuron. 1992;8:415–428. doi:10.1016/0896-6273(92)90270-N. - DOI - PubMed
    1. Brunetti-Pierri N., Doty S., Hicks J., Phan K., Mendoza-Londono R., Blazo M., Tran A., Carter S., Lewis R., Plon S., et al. Generalized metabolic bone disease in neurofibromatosis type 1. Mol. Genet. Metab. 2008;94:105–111. doi:10.1016/j.ymgme.2007.12.004. - DOI - PMC - PubMed

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