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