Elsevier

Bone

Volume 69, December 2014, Pages 30-38
Bone

Original Full Length Article
Fibulin-1 is required for bone formation and Bmp-2-mediated induction of Osterix

https://doi.org/10.1016/j.bone.2014.07.038Get rights and content

Highlights

  • Fbln1 is deposited in the ECM around bone producing cells during skull development.

  • Membranous and endochondral skull bones are reduced in Fbln1 nulls.

  • Fbln1 deficient calvariae have reduced bone volume and reduced Osterix expression.

  • Fbln1 binds Bmp-2 in in vitro binding assays.

  • Fbln1 is a positive modulator of Bmp-2-mediated induction of Osterix.

Abstract

The extracellular matrix protein Fibulin-1 (Fbln1) has been shown to be involved in numerous processes including cardiovascular and lung development. Here we have examined the role of Fbln1 in bone formation. Alizarin red staining of skulls from Fbln1-deficient mice showed reduced mineralization of both membranous and endochondral bones. MicroCT (μCT) analysis of the calvarial bones (i.e., frontal, parietal and interparietal bones collectively) indicated that bone volume in Fbln1 nulls at neonatal stage P0 were reduced by 22% (p = 0.015). Similarly, Fbln1 null frontal bones showed a 16% (p = 0.035) decrease in bone volume, with a reduction in the interfrontal bone, and a discontinuity in the leading edge of the frontal bone. To determine whether Fbln1 played a role in osteoblast differentiation during bone formation, qPCR was used to measure the effects of Fbln1 deficiency on the expression of Osterix (Osx), a transcription factor essential for osteoblast differentiation. This analysis demonstrated that Osx mRNA was significantly reduced in Fbln1-deficient calvarial bones at developmental stages E16.5 (p = 0.049) and E17.5 (p = 0.022). Furthermore, the ability of Bmp-2 to induce Osx expression was significantly diminished in Fbln1-deficient mouse embryo fibroblasts. Together, these findings indicate that Fbln1 is a new positive modulator of the formation of membranous bone and endochondral bone in the skull, acting as a positive regulator of Bmp signaling.

Introduction

Bone formation is a complex process that involves the mineralization of extracellular matrix (ECM) by osteoblasts to form bone [1] and the erosion of bone by osteoclasts to allow for growth and remodeling [2]. The formation and erosion of the mineralized matrix must be carefully coordinated and an imbalance in osteoblast and/or osteoclast activity has been shown to occur in several pathological conditions (e.g., osteoporosis, osteopetrosis) [3], [4]. The balance between osteoblast and osteoclast formation is controlled in part by ECM proteins. Bone morphogenetic protein-2 (Bmp-2) is an ECM growth factor in the TGFβ super-family [5] that stimulates mesenchymal cells to differentiate into osteoblasts [6], [7] and promotes differentiation of osteoclast precursors into mature osteoclasts [8], [9]. A relatively large number of ECM proteins negatively regulate Bmp-2-mediated osteoblast differentiation including Noggin (Nog) [10], [11], Twisted gastrulation (Twsg1) [10], [12], Connective tissue growth factor (CCN2/Ctgf) [13], Nephroblastoma overexpressed (CCN3/Nov) [14], and Fibrillin-2 (Fbn2) [15]. By contrast, a small number of ECM proteins have been defined as positive modulators of Bmp-2 signaling in bone formation (e.g., CCN4/Wisp1) [16]. Bmp-2 controls osteoblast differentiation by upregulating the osteogenic transcription factor Osterix (Osx) [7]. The importance of Osterix in osteoblast differentiation is evidenced by the absence of bone synthesis in Osterix deficient mice [17].

Fibulin-1 (Fbln1) is an ECM protein [18], [19] known to be expressed in adult bone marrow [20] and osteoblasts derived from adult bone [21]. In Fbln1 -deficient mice, which die perinatally, bone size and ossification is reduced in the skull, indicating a role for Fbln1 in bone formation [22]. Here we have performed a more detailed characterization of the consequence of Fbln1 deficiency on skull bones, investigating mechanisms whereby Fbln1 could impact the process of bone formation.

Section snippets

Fbln1-deficient mice

The studies employed two Fbln1-deficient mouse strains that have overlapping phenotypes including 1) a Fbln1 gene trap mutant [22], and 2) a strain containing targeted deletion of Fbln1 exon 1 [23]. All procedures and protocols were done in accordance with a Medical University of South Carolina IACUC approved protocol.

Histology and immunohistochemistry

P0 neonate skulls were fixed in 1× phosphate buffered saline (PBS) containing 4% paraformaldehyde for 2 h. After fixation, skulls were embedded in Optimal Cutting Temperature (OCT)

Fibulin-1-deficient skulls display reduced alizarin red staining and bone

Previous studies of mice homozygous for a gene trap insertion in the fibulin-1 gene (designated hereafter as Fbln1 nulls) have shown that Fbln1 plays a role in skull bone formation [22]. To extend on those initial findings we examined skulls from P0 wild-type and Fbln1 null neonates (P0 neonates represent the latest developmental stage that Fbln1 null mice can be obtained before lethality) using alizarin red to stain mineralized extracellular matrix. Fbln1 null mice had less alizarin red stain

Discussion

ECM proteins in bone have been shown to promote osteoprogenitor survival, proliferation and differentiation, yet these processes are poorly understood. Here we have investigated how the ECM protein Fbln1 influences bone formation, focusing on the developing skull, which displays reduced bone size and BV in Fbln1-deficient mice. The results presented here show that Fbln1 promotes formation of both the membranous and endochondral bones in the skull. Furthermore, during development, Fbln1 was

Acknowledgments

Research reported in this publication was supported in part by pilot funding from the National Institutes of Health (NIH) grants (5P20RR017696 and P30GM10331). This work was also supported by NIH grants R01HL095067, 5R21AG043718 to WSA. JLB was supported by NIH grants P30GM103342 and P20GM103499. This study used the services of the Morphology, Imaging and Instrumentation (MMI) Core, which is supported by NIH-NIGMS P30GM103342 to the South Carolina COBRE for Developmentally Based Cardiovascular

References (49)

  • M.A. Cooley et al.

    Fibulin-1 is required for morphogenesis of neural crest-derived structures

    Dev Biol

    (2008)
  • N. Kodama et al.

    A local bone anabolic effect of rhFGF2-impregnated gelatin hydrogel by promoting cell proliferation and coordinating osteoblastic differentiation

    Bone

    (2009)
  • W. Liu et al.

    Validation of a quantitative method for real time PCR kinetics

    Biochem Biophys Res Commun

    (2002)
  • T. Komori et al.

    Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts

    Cell

    (1997)
  • F. Otto et al.

    Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development

    Cell

    (1997)
  • C. Maes et al.

    Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188

    Mech Dev

    (2002)
  • M. Ishijima et al.

    Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

    Matrix Biol

    (2012)
  • W. Tang et al.

    Transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) by osteoblast-specific transcription factor Osterix (Osx) in osteoblasts

    J Biol Chem

    (2012)
  • A.B. Celil et al.

    BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways

    J Biol Chem

    (2005)
  • S. Rydziel et al.

    Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis and causes osteopenia

    J Biol Chem

    (2007)
  • I. Titorencu et al.

    Osteoblast ontogeny and implications for bone pathology: an overview

    Cell Tissue Res

    (2014)
  • M.N. Weitzmann

    The role of inflammatory cytokines, the RANKL/OPG Axis, and the immunoskeletal interface in physiological bone turnover and osteoporosis

    Scientifica (Cairo)

    (2013)
  • S.J. Lin et al.

    The structural basis of TGF-beta, bone morphogenetic protein, and activin ligand binding

    Reproduction

    (2006)
  • L. Pham et al.

    Bone morphogenetic protein 2 signaling in osteoclasts is negatively regulated by the BMP antagonist, twisted gastrulation

    J Cell Biochem

    (2011)
  • Cited by (26)

    • cBiT: A transcriptomics database for innovative biomaterial engineering

      2017, Biomaterials
      Citation Excerpt :

      These genes include FBLN1 (Fibulin 1), HBEGF (Heparin-binding EGF-like growth factor), SPP1 (Osteopontin), PLAUS (Plasminogen activator, urokinase receptor), CXCL8 (C-X-C Motif Chemokine Ligand 8), and ASS1 (Argininosuccinate synthase 1). FBLN1, HBEGF, SPP1, PLAUS, and CXCL8 indeed have a clear relation with either osteogenesis or osteoclastogenesis [35–39]. This relationship is less clear for the ASS1 gene and therefore makes it an interesting candidate for further analysis.

    • Fibulins and matrilins are novel structural components of the periodontium in the mouse

      2017, Archives of Oral Biology
      Citation Excerpt :

      In the alveolar bone, all fibulins and matrilin-1 and -2 were expressed in pericellular regions of osteocytes suggesting a participation of the proteins in the mineralization process of the bone ECM. For fibulin-1, an involvement in the craniofacial bone formation has been described (Cooley et al., 2014); fibulin-5 is indispensible for the regulation of mesenchymal cell proliferation in premaxillary bone sutures (Noda, Nakamura, & Komatsu, 2015). The present results indicate a role for fibulins in the adult murine bone beyond craniofacial development.

    • Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing

      2017, Developmental Biology
      Citation Excerpt :

      Two other fibulin family genes have been reported to regulate TGF-β family signaling. Fibulin-1 regulates BMP signaling through direct binding to BMP-2 (Cooley et al., 2014), whereas Fibulin-3 inhibits TGF-β1 signaling by interacting with TβRI/ALK-5 to prevent formation of a functional ligand-receptor complex (Tian et al., 2015). Fibulin-4 mutations are associated with patients with aortic aneurysms, arterial tortuosity and stenosis.

    • Fibulin-5 deficiency causes developmental defect of premaxillary bone in mice

      2015, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Among seven fibulin family proteins, fibulin-1 and fibulin-5 are reported to be expressed in mouse neural crest derivatives, including craniofacial mesenchymal tissues [4,5,11,12]. While fibulin-1 is critical for cranial bone formation [13,14], the importance of fibulin-5 in craniofacial development has not been unraveled. We report here that fibulin-5 is localized in postnatal craniofacial sutures, and contributes to facial skeletal development.

    View all citing articles on Scopus
    View full text