A low bone mass phenotype in the murine model with bone conditional inactivation of TMEM38B

Osteogenesis imperfecta (OI) or brittle-bone disease is a group of rare collagenopathies characterized by reduced bone mineral density and increased bone fragility. The autosomal recessive OI type XIV (OMIM #615066) is caused by mutations in the TMEM38B gene encoding for the trimeric intracellular cation channel B (TRIC-B), an ER channel specific for K+ ions necessary as counter ions for intracellular calcium flux. The molecular basis of the OI type XIV bone phenotype is still puzzling the field. To study the role of the TRIC-B channel, a TRIC-B knock-out mouse was generated but resulted in a lethal phenotype due to respiratory failure. The aim of my thesis was to evaluate the TRIC-B function specifically in the bone tissue. To this purpose, we generated with a embryonic stem cells (ESC) gene targeting approach a Tmem38b osteoblast specific conditional knock-out (Runx2Cre;Tmem38bfl/fl). The analyses of the skeleton revealed the presence of bone deformations and calli in the long bones of mutant mice and a mineralization delay in vertebrae. Also, a growth delay was found in mutant mice, in agreement with OI human features. Micro computed tomography analysis of the bone geometrical properties revealed the presence of an osteoporotic phenotype affecting both the cortical and the trabecular bone, thus revealing the primary function of TRIC-B for the development of the bone tissue. The impact of loss of Tmem38b function on osteoblasts activity was then investigated in vitro in primary calvarial osteoblasts. Mineralization and alkaline phosphatase activity were decreased in mutant osteoblasts and the analyses of osteoblastogenic genes expression revealed a delay in osteoblasts differentiation. Importantly, in mutant osteoblasts we detected the presence of large vacuoles and enlarged ER cisternae, as well as the presence of intracellular accumulation of misfolded material. In conclusion, the Runx2Cre;Tmem38bfl/fl mouse represents a valid model of OI type XIV that will allow to study the TRIC-B role in calcium homeostasis and to understand how a defective ER calcium flux dysregulates collagen synthesis resulting in the OI phenotype.