title: Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models.
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 normal osteocyte 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.
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité, Universitätsmedizin Berlin, Berlin, Germany; FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany. Electronic address: jirko.kuehnisch@gmx.de.
authors.forename: Jirko
authors.initials: J
authors.lastname: Kühnisch
authors.affiliation: Department of Biomaterials, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany; Department of Chemistry, École Normale Superiéure, 24 rue Lhomond, Paris 75005, France.
authors.forename: Jong
authors.initials: J
authors.lastname: Seto
authors.affiliation: Department 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.
authors.forename: Claudia
authors.initials: C
authors.lastname: Lange
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité, Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Stumpp
authors.affiliation: FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Karolina
authors.initials: K
authors.lastname: Kobus
authors.affiliation: FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Grohmann
authors.affiliation: Department of Medicine, Pharmacology and Cancer Biology, Center for Bone Biology, Vanderbilt University Medical Center, Nashville TN, USA.
authors.forename: Florent
authors.initials: F
authors.lastname: Elefteriou
authors.affiliation: Department of Biomaterials, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany; Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Fratzl
authors.affiliation: Institute 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.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mundlos
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité, Universitätsmedizin Berlin, Berlin, Germany; FG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany. Electronic address: kolanshy@molgen.mpg.de.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Kolanczyk
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic / Q000235:genetics; D048430:Cell Shape; D002470:Cell Survival; D004734:Energy Metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation; D006811:Humerus / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D023421:Models, Animal; D025542:Neurofibromin 1 / Q000172:deficiency / Q000378:metabolism*; D010011:Osteocytes / Q000378:metabolism* / Q000473:pathology* / Q000648:ultrastructure; D013314:Stress, Mechanical; D014158:Transcription, Genetic
chemical_list: D025542:Neurofibromin 1
keywords: Bone; MAPK; NF1; neurofibromin; osteocyte