Abstract
BRIL (bone restricted ifitm-like; also known as IFITM5) is a transmembrane protein expressed in osteoblasts. Although its role in skeletal development and homeostasis is unknown, mutations in BRIL result in rare dominant forms of osteogenesis imperfecta. The pathogenic mechanism has been proposed to be a gain-of or neomorphic function. To understand the function of BRIL and its OI type V mutant (MALEP BRIL) and whether they could activate signaling pathways in osteoblasts, we performed a luciferase reporter assay screen based on the activity of 26 transcription factors. When overexpressed in MC3T3-E1 and MLO-A5 cells, the MALEP BRIL activated the reporters dependent on MEF2, NFATc, and NR4A significantly more. Additional co-transfection experiments with MEF2C and NFATc1 and a number of their modulators (HDAC4, calcineurin, RCAN, FK506) confirmed the additive or synergistic activation of the pathways by MALEP, and suggested a coordinated regulation involving calcineurin. Endogenous levels of Nr4a members, as well as Ptgs2, were upregulated by MALEP BRIL. Y2H and co-immunoprecipitation indicated that BRIL interacted with CAML, but its contribution as the most upstream stimulator of the Ca2+-calcineurin-MEF2/NFATc cascade was not confirmed convincingly. Altogether the data presented provide the first ever readout to monitor for BRIL activity and suggest a potential gain-of-function causative effect for MALEP BRIL in OI type V, leading to perturbed signaling events and gene expression.
Highlights
Published: 15 February 2022Osteogenesis imperfecta (OI) is a rare heritable connective tissue disorder mainly characterized by bone fragility, low bone mass, and short stature
BRIL pathologic importance has been exemplified by a distinct set of mutations in dominant OI types
In the current study we identified that specific intracellular pathways are activated by the wild type (WT) and mutant MALEP BRIL
Summary
Published: 15 February 2022Osteogenesis imperfecta (OI) is a rare heritable connective tissue disorder mainly characterized by bone fragility, low bone mass, and short stature. The most severely affected patients suffer from debilitating skeletal deformities and multiple fractures [1–3]. 85% of OI patients have autosomal dominant mutations in COL1A1 and COL1A2 [1–3]. The only dominant OI types not caused by mutations in COL1 genes harbor mutations in the interferon induced transmembrane protein 5 gene (IFITM5 hereafter designated BRIL (bone-restricted Ifitm-like)). There have been five genetic variants in BRIL reported so far: c.-14C>T (p.M1ext-5) [4,5] in OI type V; c.119C>T (p.S40L) [6,7] and c.119C>G (p.S40W) [8,9] in atypical type VI; and the following two most recent that have not yet been classified (c.143A>G (p.N48S) [10] and c.-9C > A (p.M1ext-3) [11]). OI type V is estimated to be the second most prevalent form of OI [12], with about
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