Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV

Abstract

Osteogenesis imperfecta (OI) type XIV is a rare recessive bone disorder characterized by variable degree of severity associated to osteopenia. It is caused by mutations in TMEM38B encoding for the trimeric intracellular cation channel TRIC-B, specific for potassium and ubiquitously present in the endoplasmic reticulum (ER) membrane. OI type XIV molecular basis is largely unknown and, due to the rarity of the disease, the availability of patients’ osteoblasts is challenging. Thus, CRISPR/Cas9 was used to knock out (KO) TMEM38B in the human Foetal Osteoblast hFOB 1.19 to obtain an OI type XIV model. CRISPR/Cas9 is a powerful technology to generate in vitro and in vivo models for heritable disorders. Its limited cost and ease of use make this technique widely applicable in most laboratories. Nevertheless, to fully take advantage of this approach, it is important to be aware of its strengths and limitations. Three gRNAs were used and several KO clones lacking the expression of TRIC-B were obtained. Few clones were validated as good models for the disease since they reproduce the altered ER calcium flux, collagen I structure and impaired secretion and osteoblastic markers expression detected in patients’ cells. Impaired proliferation and mineralization in KO clones unveiled the relevance of TRIC-B in osteoblasts functionality.

Emerging Landscape of Osteogenesis Imperfecta Pathogenesis and Therapeutic Approaches

“…Defects in the TMEM38B-encoded TRICB-type trimeric intracellular cation channel B (TRICB, also known as TM38B ) have been identified in patients with moderate OI. , Recent studies indicate that TRIC-B cation-specific channels in the ER membrane, encoded by TMEM38B, are implicated in the OI phenotype . Knockout mice lacking TMEM38B showed decreased mortality and bone volume during the embryonic stage .…”

Zebrafish Tric-b is required for skeletal development and bone cells differentiation

“…Osteoclasts are also impaired, since their number and activity are reduced ( 16 ). In immortalized human Foetal Osteoblasts (hFOB) knock-out for TMEM38B a decreased proliferation and mineralization have been recently demonstrated ( 19 ).…”

Intracellular Ca2+ signalling: unexpected new roles for the usual suspect

“…In both excitable and non-excitable cells, extracellular Ca 2+ entry is further mediated by the Transient Receptor Potential (TRP) family of non-selective cation channels, most of which are polymodal Ca 2+ -permeable channels able to sense chemical, thermal and mechanical signals and thereby execute the most appropriate cellular response ( Curcic et al, 2019 ; Vangeel and Voets, 2019 ; Diver et al, 2022 ). The advent of novel high-speed, 2D and 3D time-lapse imaging techniques, single-wavelength and genetic Ca 2+ indicators, as well as the development of novel genetic engineering tools to manipulate single cells and whole animals, has shed novel light on the regulation of cellular activity by the Ca 2+ handling machinery ( Lim et al, 2016a ; Bagur and Hajnoczky, 2017 ; Tapella et al, 2020 ; Berra-Romani et al, 2021 ; Leoni et al, 2021 ; Longden et al, 2021 ; Marta et al, 2022 ). For instance, it has been recognized that ER cisternae may establish dynamic contacts with other intracellular organelles, such as mitochondria ( Csordas et al, 2010 ; Csordas et al, 2018 ; Bartok et al, 2019 ; Lim et al, 2021a ; Sanchez-Vazquez et al, 2023 ) and lysosomes ( Kilpatrick et al, 2013 ; Atakpa et al, 2018 ; Faris et al, 2022 ), to shape intracellular Ca 2+ signals.…”