Marie-Hélène Gaumond

Bril: A novel bone-specific modulator of mineralization

In the course of attempting to define the bone “secretome” using a signal‐trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm‐like protein (Bril). Bril encodes a 14.8‐kDa 134 amino acid protein with two transmembrane domains. Northern blot analysis showed bone‐specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus‐mediated Bril overexpression and lentivirus‐mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose‐dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype.

Although a number of secreted factors have been demonstrated to be bone regulators, none of these are unique to bone. Using a viral-based signal-trap strategy we have identified a novel gene we have termed “osteocrin.” A 1280-bp mRNA encodes osteocrin producing a mature protein of 103 amino acids with a molecular mass of 11.4 kDa. Osteocrin shows no homology with any known gene except for two conserved sequence motifs reminiscent of dibasic cleavage sites found in peptide hormone precursors. Immunofluorescence and Western blot analysis confirmed the secretory nature of osteocrin. Two protein species were identified in the medium of cells overexpressing osteocrin, a full-length 11.4 kDa species and a processed ∼5 kDa species. Mutation of the 76KKKR79 dibasic cleavage site abolished the appearance of this smaller osteocrin fragment. By in situ hybridization in mouse embryos, osteocrin was expressed specifically in Cbfa-1-positive, osteocalcin-negative osteoblasts. Immunohistochemistry on adult mouse bone showed osteocrin localization in osteoblasts and young osteocytes. By Northern blot analysis, osteocrin expression was only detected in bone, expression peaking just after birth and decreasing markedly with age. In primary osteoblastic cell cultures osteocrin expression coincided with matrix formation then decreased in very mature cultures. Treatment of cultures with 1,25-dihydroxyvitamin D3 resulted in a rapid dose-dependent down-regulation of osteocrin expression, suggesting direct regulation. Chronic treatment of primary cultures with osteocrin-conditioned media inhibited mineralization and reduced osteocalcin and alkaline phosphatase expression. These results suggest that osteocrin represents a novel, unique vitamin D-regulated bone-specific protein that appears to act as a soluble osteoblast regulator.

Topological Mapping of BRIL Reveals a Type II Orientation and Effects of Osteogenesis Imperfecta Mutations on Its Cellular Destination

BRIL/IFITM5 is a membrane protein present almost exclusively in osteoblasts, which is believed to adopt a type III (N‐out/C‐out) topology. Mutations in IFITM5 cause OI type V, but the characteristics of the mutant protein and the mechanism involved are still unknown. The purpose of the current study was to re‐assess the topology, localization, and biochemical properties of BRIL and compare it to the OI type V mutant in MC3T3 osteoblasts. Immunofluorescence labeling was performed with antibodies directed against BRIL N‐ or C‐terminus. In intact cells, BRIL labeling was conspicuously detected at the plasma membrane only with the anti‐C antibody. Detection of BRIL N‐terminus was only possible after cell permeabilization, revealing both plasma membrane and Golgi labeling. Trypsinization of live cells expressing BRIL only cleaved off the C‐terminus, confirming that it is a type II protein and that its N‐terminus is intracellular. A truncated form of BRIL lacking the last 18 residues did not appear to affect localization, whereas mutation of a single leucine to arginine within the transmembrane segment abolished plasma membrane targeting. BRIL is first targeted to the endoplasmic reticulum as the entry point to the secretory pathway and rapidly traffics to the Golgi via a COPII‐dependent pathway. BRIL was found to be palmitoylated and two conserved cysteine residues (C52 and C53) were critical for targeting to the plasma membrane. The OI type V mutant BRIL, having a five residue extension (MALEP) at its N‐terminus, presented with exactly the same topological and biochemical characteristics as wild type BRIL. In contrast, the S42 > L mutant BRIL was trapped intracellularly in the Golgi. BRIL proteins and transcripts were equally detected in bone from a patient with OI type V, suggesting that the cause of the disease is a gain of function mediated by a faulty intracellular activity of the mutant BRIL.

Regulation of the Bone-restricted IFITM-like (Bril) Gene Transcription by Sp and Gli Family Members and CpG Methylation

Background: BRIL is a bone-specific membrane protein that is involved in osteogenesis imperfecta type V. Results: Bril transcription is activated by Sp1, Sp3, OSX, and GLI2 and by CpG demethylation. Conclusion: Regulation of Bril involves trans-acting factors integrating at conserved promoter elements and epigenetic modifications. Significance: Identification of the mechanisms governing Bril transcription is important to understand its role in skeletal biology. Bril encodes a small membrane protein present in osteoblasts. In humans, a single recurrent mutation in the 5′-UTR of BRIL causes osteogenesis imperfecta type V. The exact function of BRIL and the mechanism by which it contributes to disease are still unknown. The goal of the current study was to characterize the mechanisms governing Bril transcription in humans, rats, and mice. In the three species, as detected by luciferase reporter assays in UMR106 cells, we found that most of the base-line regulatory activity was localized within ∼250 bp upstream of the coding ATG. Co-transfection experiments indicated that Sp1 and Sp3 were potent inducers of the promoter activity, through the binding of several GC-rich boxes. Osterix was a weak activator but acted cooperatively with Sp1 and GLI2 to synergistically induce the BRIL promoter. GLI2, a mediator of hedgehog signaling pathway, was also a potent activator of BRIL through a single GLI binding site. Correspondingly, agonists of the hedgehog pathway (purmorphamine and Indian hedgehog) in MC3T3 osteoblasts led to increased BRIL levels. The BRIL promoter activity was also found to be negatively modulated through two different mechanisms. First, the ZFP354C zinc finger protein repressed basal and Sp1-induced activity. Second, CpG methylation of the promoter region correlated with an inactive state and prevented Sp1 activation. The data provide the very first analyses of the cis- and trans-acting factors regulating Bril transcription. They revealed key roles for the Sp members and GLI2 that possibly cooperate to activate Bril when the promoter becomes demethylated.

The Potential Use of Nanoparticles for Noggin siRNA Delivery toAccelerate Bone Formation in Distraction Osteogenesis

In bone research and orthopedics, RNA-interference (RNAi) and nanotechnology can be applied to a vast array of conditions where bone formation needs to be enhanced, providing new options to treat old problems, from osteoporosis and bone tumors to nonunion and critical size defects. The need for new therapeutic tools to improve bone regeneration is evident, mainly in more complex orthopedic surgical procedures. Distraction Osteogenesis (DO) is a unique surgical technique that stimulates bone formation, most frequently used to promote limb lengthening through slow and progressive distraction after osteotomy. The great challenge is to reduce the consolidation phase and prevent the complications related to the maintenance of the external fixator for a long period. Bone Morphogenetic Proteins (BMPs) have been used as potent osteoinductive growth factors, but we considered manipulating the BMP-antagonist NOGGIN through RNA silencing to increase endogenous BMPs levels, as an option for the controversial use of exogenous BMPs. For RNAi-based therapies, the greatest challenge over the past 16 years has been to achieve safe, effective and specific delivery to the target tissue. In-vivo studies employed a variety of delivery methods aiming for the ultimate clinical translational application for RNAi technology, but RNAi drugs are still undergoing clinical trials for approval by the US Food and Drug Administration (FDA). This review considers the application of nanotechnology to potentially address the difficulties of delivering silencing RNAs in vivo and in clinical applications, and presents preliminary studies about downregulation of the BMP-antagonist NOGGIN through RNAi in rat bone cells and delivery of siRNA sequences to bone tissue by nanoparticles.

The osteogenic cell surface marker BRIL/IFITM5 is dispensable for bone development and homeostasis in mice

BRIL (bone-restricted IFITM-like), is a short transmembrane protein expressed almost exclusively in osteoblasts. Although much is known about its bone-restricted gene expression pattern and protein biochemical and topological features, little information is available for BRIL physiological function. Two autosomal dominant forms of osteogenesis imperfecta (OI) are caused by distinct, but recurrent mutations in the BRIL gene. Yet, the underlying mechanisms by which those mutations lead to OI are still poorly understood. A previous report indicated that BRIL knockout (KO) mice had bone deformities, shortened long bones, and reproductive problems. Here we generated and systematically analyzed the skeletal phenotype of a new global Bril KO/LacZ knockin mouse model. KO mice reproduced and thrived normally up to 12 month of age. The skeletal phenotype of KO and WT littermates was assessed at embryonic (E13.5 to E18.5) and postnatal (2 days, 3 weeks, 3 months and 8 months) time-points. Embryos from E13.5 through to E18.5 showed significant X-Gal staining in all skeletal elements without any apparent patterning anomalies. Although bone deformities were never observed at any postnatal ages, minor and transient differences were noted in terms of bone length and static uCT parameters, but not systematically across all ages and genders. These changes, however, were not accompanied by significant alteration in bone material properties as assessed by a 3-point bending test. In addition, no changes were detected in circulating serum markers of bone turnover (P1NP, CTX-I, and osteocalcin). Gene expression monitoring also revealed no major impact of the loss of BRIL. Further, when mice were challenged with a surgically-induced fracture in tibia, bones repaired equally well in the KO mice as compared to WT. Finally, we showed that BRIL C-terminus is not a bona fide binding site for calcium. In conclusion, our in depth analysis suggest that skeletal patterning, bone mass accrual and remodeling in mice proceeded independent of BRIL.