THU407 CRISPR/Cas9-based Knockout Of CFTR Alters Mechano-sensitivity And Proteomic Profile In An Osteocyte Model

Disclosure: W. Du: None. J. Chen: None. Y. Ruan: None. Osteocytes, the most abundant bone cell type with recognized endocrine functions, play essential roles in mechano-signal transduction in the skeletal system required for bone modelling/remodelling and mineral homeostasis, although the underlying molecular mechanisms are not fully understood. Bone problems are present in cystic fibrosis (CF), a genetic disease resulted from mutations in CF transmembrane conductance regulator (CFTR), which is a Cl(−) channel gene recently revealed to be expressed in osteocytes with its exact roles largely unexplored. In the present study, CRISPR/Cas9 technology was used to knock out CFTR (CFTR-KO) in MLO-Y4, a commonly used osteocyte model, which resulted in a significant decrease (by 80.7 ± 7.4%) in shear force-induced Ca(2+) oscillations (measured by Fura-2 imaging) as compared to wild-type (WT) cells (n = 205 (WT) and 114 (KO) cells, t-test, p < 0.001). However, the CFTR-KO cells did not show, in comparison with WT cells, any reduction in expressing (measured by quantitative PCR, n = 5) Piezo1 or TRPV4, two reported key mechano-sensitive Ca(2+) channels in osteocytes, nor change in cell membrane stiffness (measured by atom force microscope, n = 58 (WT) and 64 (KO) cells, t-test, p > 0.05). Moreover, the shear-force-stimulated Ca(2+) oscillations in WT cells were observed to be dependent on extracellular Cl(−), which together suggested possibly a direct role of CFTR, through its Cl(-) channel function, in osteocyte mechano-sensitivity. We next performed proteomic analysis by mass spectrometry detection of whole-cell protein extracts from the MLO-Y4 cells, which revealed downregulation of genes related to focal adhesion, gap junction and actin cytoskeleton regulation in CFTR-KO cells. These results, therefore, have suggested a crucial involvement of CFTR in osteocyte function, providing new insights into mechanistic understanding of the mechano-transduction in the skeletal system. This work was supported in part by Health and Medical Research Fund of Hong Kong (No.18191361), Theme-based Research Scheme of Hong Kong (No. T13-402/17N), and Areas of Excellence Scheme of Hong Kong (AoE/M-402/20). Presentation: Thursday, June 15, 2023