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Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models

Significance Statement

Neurofibromin, which is mutated in the benign tumor entity Neurofibromatosis type 1, has been identified as a critical regulator of osteoblast differentiation. Osteoblast specific deactivation of neurofibromin in mice results in a high bone mass phenotype and hyperosteoidosis. Osteocytes are the most abundant bone cell type and determine bone mass by integration of mechanical and endocrine signals. In this study, we investigate the impact of neurofibromin on osteocyte differentiation in conditional mouse models. Osteocyte lacunae are significantly enlarged after loss of neurofibromin. In addition, the dendritic network of these osteocytes appears severely disordered. On the molecular level, osteocytes demonstrate hyperactivate MAPK signaling. We further detected down-regulation of genes that are involved in mechanical sensing and organic matrix formation. Genes representing tumor vessel abundance and cellular lipid metabolism were upregulated. We conclude that neurofibromin determines both osteoblast and osteocyte differentiation. Thus, neurofibromin likely regulates mechanical sensing, bone matrix composition and mechanical resistance of bone tissue.

Figure Legend

Ablation of neurofibromin in mesenchymal progenitor cells affects osteoblast and osteocyte differentiation. Bone mass is determined by osteoblasts, osteoclasts and osteocytes. Ablation of neurofibromin in pre-osteoblasts (Nf1Col1) results in a high bone mass phenotype with hyperosteoidosis. Loss of neurofibromin in mesenchymal progenitor cells (Nf1Prx1) produces a complex phenotype characterized by low bone mass, hyperosteoidosis, increased micro-porosity (Ot.), macro-porotic mineralization lesions, and persistence of blood vessels. Moreover, loss of neurofibromin causes hyperactive MAPK signaling inducing defective inorganic and organic bone matrix formation especially in proximity of blood vessels. Thus, neurofibromin critically regulates osteoblast and osteocyte differentiation.

Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models

 

 

 

 

 

 

 

 

Journal Reference

Kühnisch J1, Seto J2, Lange C3, Stumpp S4, Kobus K5, Grohmann J5, Elefteriou F6, Fratzl P7, Mundlos S8, Kolanczyk M9. Bone. 2014;66:155-62.

Show Affiliations

1Institute for Medical Genetics and Human Genetics, Charité, Universitätsmedizin Berlin, Berlin, Germany; FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany. Electronic address: [email protected]

2Department of Biomaterials, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany; Department of Chemistry, École Normale Superiéure, 24 rue Lhomond, Paris 75005, France.

3Department of Biomaterials, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany; Institut für Physiologische Chemie, MTZ, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.and

4Institute for Medical Genetics and Human Genetics, Charité, Universitätsmedizin Berlin, Berlin, Germany.and

5FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany.

6Department of Medicine, Pharmacology and Cancer Biology, Center for Bone Biology, Vanderbilt University Medical Center, Nashville TN, USA.and

7Department of Biomaterials, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany; Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.

8Institute for Medical Genetics and Human Genetics, Charité, Universitätsmedizin Berlin, Berlin, Germany; FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany; Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.

9Institute for Medical Genetics and Human Genetics, Charité, Universitätsmedizin Berlin, Berlin, Germany; FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany. Electronic address: [email protected]

 

Abstract

Neurofibromin has been identified as a critical regulator of osteoblast differentiation. Osteoblast specific inactivation of neurofibromin in mice results in a high bone mass phenotype and hyperosteoidosis. Here, we show that inactivation of the Nf1 gene also impairs osteocyte development. We analyzed cortical bone tissue in two conditional  mouse  models, Nf1Prx1 and Nf1Col1, for morphological and molecular effects. Backscattered electron microscopy revealed significantly enlarged osteocyte lacunae in Nf1Prx1 and Nf1Col1 mice (level E2: ctrl=1.90±0.52%,Nf1Prx1=3.40±0.95%; ctrl 1.60±0.47%, Nf1Col1 2.46±0.91%). Moreover, the osteocyte lacunae appeared misshaped in Nf1Prx1 and Nf1Col1 mice as indicated by increased Feret ratios. Strongest osteocyte and dendritic network disorganization was observed in proximity of muscle attachment sites in Nf1Prx1 humeri. In contrast to control cells, Nf1Prx1 osteocytes contained abundant cytosolic vacuoles and accumulated immature organic matrix within the perilacunar space, a phenotype reminiscent of the hyperosteoidosis shown Nf1 deficient mice. Cortical bone lysates further revealed approx. twofold upregulated MAPK signalling in osteocytes of Nf1Prx1 mice. This was associated with transcriptional downregulation of collagens and genes involved in mechanical sensing in Nf1Prx1 and Nf1Col1 bone tissue. In contrast, matrix gla protein (MGP), phosphate regulating endopeptidase homolog, X-linked (PHEX), and genes involved in lipid metabolism were upregulated. In line with previously described hyperactivation of Nf1 deficient osteoblasts, systemic plasma levels of the bone formation markers osteocalcin (OCN) and procollagen typ I N-propeptide (PINP) were approx. twofold increased in Nf1Prx1 mice. Histochemical and molecular analysis ascertained that osteocytes in Nf1Prx1 cortical bone were viable and did not undergo apoptosis or autophagy. We conclude that loss of neurofibromin is not only critical for osteoblasts but also hinders normalosteocyte development. These findings expand the effect of neurofibromin onto yet another cell type where it is likely involved in the regulation of mechanical sensing, bone matrix composition and mechanical resistance of bone tissue.

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