Abstract
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.
Highlights
Real-time quantitative polymerase chain reaction (RT-qPCR) analyses confirmed that the gene had been successfully inactivated (Fig 1C), as no transcript was detected in 6w old Bril KO humeri
The full length cDNA cloning for Bril was first reported from primitive hemopoietic cells differentiated from ES cell embryonic bodies [30]
It was subsequently identified as part of the Ifitm family cluster [31]
Summary
Bone-restricted IFITM-like (BRIL), known as interferon-induced transmembrane protein 5 (IFITM5), is a small 132 amino acid membrane protein expressed in osteoblasts. Characteristics and properties of BRIL make it highly unique from other family members, including the bone-specific expression profile of the protein [1, 2] and gene regulation by mechanisms not involving induction by interferons [5, 6]. The OI disease causing mutations in Bril are believed to result in contrasting neomorphic functions [3, 16], typified on one hand by ectopic mineralization in type V, and on the other by decreased mineralized osteoid in the atypical type VI These differing human conditions would not help clarify the underlying mechanism of action and normal activity carried out by BRIL. Studies by Lietman et al [16] have demonstrated that osteoblast-specific overexpression of BRIL in a transgenic mouse model, under regulation of the collagen type I promoter, resulted in an absence of phenotypic consequences on the skeleton. Our data indicate that the absence of BRIL does not significantly impact skeletal integrity in vivo in mice
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