Elsevier

Bone

Volume 66, September 2014, Pages 155-162
Bone

Original Full Length Article
Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models

https://doi.org/10.1016/j.bone.2014.06.012Get rights and content

Highlights

  • Nf1Prx1 and Nf1Col1 mice show enlarged osteocyte lacunae and disturbed canalicular network organization in cortical bone.

  • Osteocytes in Nf1Prx1 bone are viable and do not show autophagy.

  • Nf1 deficiency increases Mgp, Phex and Tem7 expression.

  • Transcriptional profiling reveals molecular changes in cytoskeletal function, energy metabolism and ECM properties.

  • Nf1 is critical for osteoblast to osteocyte transition.

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.

Introduction

Osteocytes constitute 90% of all cells of adult cortical bone [1]. Embedded in the mineralized matrix, osteocytes communicate with one another through a network of dendritic processes, the canalicular network, and are capable of regulating local bone turnover by recruiting osteoclasts and osteoblasts [1], [2], [3]. Osteocytes are formed by differentiation of mesenchymal progenitor cells into osteoblasts and their subsequent incorporation into the bone matrix [1]. During differentiation osteocytes acquire specific morphological properties including dendritic processes and small cell body volume. This specific adaptation of morphology and ultrastructure allows osteocytes to perform their function as mechanosensory cell embedded within the mineralized bone. The osteocyte canalicular system is thought to facilitate the mechanosensory function by providing attachment sites for the cell processes, which are the main sites of the mechanical stimulation [4].

Osteocytes respond to mechanical stimulation with rapid synthesis of nitric oxide (NO) and prostaglandins [1]. This in turn leads to downregulation of WNT pathway inhibitors SOST and DKK1, which constitutes a bone-anabolic signal [5]. Osteocytes actively remodel their perilacunar matrix with help of metalloproteinases thus regulating phosphate and calcium availability [6]. Both osteoblasts as well as the early osteocytes regulate the degree of matrix mineralization in their surrounding with help of SIBLING family of proteins: osteopontin (OPN), dentin matrix protein 1 (DMP1) and matrix extracellular phosphoglycoprotein (MEPE) as well as with Matrix Gla Protein (MGP) [7]. While OPN, MEPE and MGP inhibit mineralization, DMP1 is a pro-mineralization factor [8]. Inactivating mutations of DMP1 or endopeptidase PHEX result in bone mineralization defects that are associated with increased FGF23 expression in osteocytes [9]. FGF23 is an endocrine regulator of systemic phosphate metabolism that is mainly synthesized by osteocytes [8]. Mechanical stimulation ensures osteocyte viability by preventing apoptosis [10]. This is achieved at least in part by the activation of focal adhesion kinases (FAKs) and extracellular signals-regulated kinases (ERKs) [10]. In addition, ERK signalling is critical for Dmp1 expression and osteocyte differentiation [11]. Apart from apoptosis osteocytes can undergo a process aimed at self-preservation called autophagy, which is induced by glucocorticoids [12].

Biallelic inactivation of the Nf1 gene results in a profound skeletal pathology in patients with Neurofibromatosis type 1 (NF1) and corresponding conditional mouse models [13], [14], [15]. Inactivation of Nf1 specifically in osteoblasts (Nf1Col1 mice) causes increased collagen synthesis, but inhibits bone mineralization resulting in osteoidosis [13], [14]. Osteoblast dysfunction is therefore an important factor contributing deterioration of bone material properties [13]. We have shown that ablation of Nf1 in undifferentiated limb mesenchyme and derivative tissues produces in Nf1Prx1 mice a severe bone phenotype characterized by increase of micro-porosity, hypomineralization, a generalized defect of organic matrix formation, and persistence of ectopic blood vessels that are associated with localized macro-porotic bone lesions [16]. This phenotype overlaps with findings made in NF1 patient material suggesting a complex mechanism leading to bone fragility in NF1 [16]. Increased micro-porosity and reduced organic matrix quality are likely caused by defective osteocyte development. In order to further characterize the role of osteocytes in NF1 bone dysplasia, we now focused on the analysis of molecular and histological aspects of the pathology in Nf1 deficient murine cortical bone. We hypothesize that Nf1 affects osteocyte differentiation.

Section snippets

Mouse breeding and genotyping

Nf1Prx1 and Nf1Col1 mice were bred and genotyped as described previously [13], [14], [17]. All experimental procedures were approved by the ‘Landesamt für Gesundheitsschutz und Technische Sicherheit (LaGeTSi), Berlin, Germany (protocol number ZH 120) and the Institutional Animal Care and Use Committee (IACUC) at the Vanderbilt University Medical Center (protocol number M/06/508).

Protein and mRNA analysis

Cortical bone was mechanically cleaned from adjoining muscle and connective tissue. Bone marrow was removed by a

Altered osteocyte morphology in Nf1Prx1 and Nf1Col1 humerus cortex

Nf1 was inactivated in the mesenchymal lineage (Nf1Prx1) and in pre-osteoblasts (Nf1Col1) as previously described [16]. In order to analyse the morphology of osteocyte (Ot.) lacunae, we performed backscattered electron microscopy (BSE) on methacrylate embedded humeri of three month old mice. Cortical bone sections were analysed along the proximo-distal axis. Three regions of interest (ROI E1-E3) were selected for qualitative and quantitative analysis. BSE revealed striking qualitative changes

Discussion

Deregulation of osteoblast and osteoclast activity in Nf1 deficient bone yield a high-turnover phenotype, reduced bone mass and low bone mineral content [13], [14], [29], [30]. Detailed structural analysis revealed increased micro-porosity in Nf1Col1 and Nf1Prx1 cortical bones, which was due to increased osteocyte lacunae size but not osteocyte number [16]. However, no histological or molecular data were available to support the role of osteocytes in the NF1 bone pathology. Here, we show that

Conflict of interest

Authors have no competing interests to declare.

Acknowledgments

We thank Petra Schrade and Sebastian Bachmann from the electron microscopy core facility of the Charité - Universitätsmedizin Berlin for excellent assistance. We also thank Monika Osswald for excellent technical assistance.

Authors´ roles: Study design: JK, FE, PF, SM, MK. Performed research: JK, JS, CL, SS, KK, JG, MK. Analyzed data: JK, MK. Wrote the manuscript: JK, MK. All authors read and approved the manuscript.

References (41)

  • M.M. Thi et al.

    Mechanosensory responses of osteocytes to physiological forces occur along processes and not cell body and require alphaVbeta3 integrin

    Proc Natl Acad Sci U S A

    (2013)
  • C. Galli et al.

    Osteocytes and WNT: the mechanical control of bone formation

    J Dent Res

    (2010)
  • K. Holmbeck et al.

    The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone

    J Cell Sci

    (2005)
  • J.Q. Feng et al.

    Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism

    Nat Genet

    (2006)
  • A. Martin et al.

    Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway involving FGF receptor (FGFR) signaling

    FASEB J

    (2011)
  • L.I. Plotkin et al.

    Mechanical stimulation prevents osteocyte apoptosis: requirement of integrins, Src kinases, and ERKs

    Am J Physiol Cell Physiol

    (2005)
  • A. Kyono et al.

    FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation

    J Bone Miner Metab

    (2012)
  • X. Xia et al.

    Glucocorticoid-induced autophagy in osteocytes

    J Bone Miner Res

    (2010)
  • M. Kolanczyk et al.

    Multiple roles for neurofibromin in skeletal development and growth

    Hum Mol Genet

    (2007)
  • J. Kuhnisch et al.

    Multiscale, Converging Defects of Macro-Porosity, Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

    PLoS One

    (2014)
  • Cited by (5)

    View full text