Role of Piezo1-mediated mechanotransduction in osteocyte differentiation

Mechanical force is a fundamental regulator of bone development and homeostasis. Mechanosensitive osteocytes are the most abundant bone cells that form interconnected dendrites to respond to mechanical stimuli and interact with the bone-forming osteoblasts and the bone-remodeling osteoclasts. However, the molecular mechanisms underlying osteocyte maturation and dendrite formation remain unclear. By generating a Piezo1 "knock-out" osteocyte cell line, we identified a key role of Piezo1-mediated mechanotransduction in osteocyte differentiation. QRT-PCR analysis revealed delayed osteocyte differentiation in the Piezo1 KO cells relative to WT cells. By performing bulk RNA sequencing of WT and Piezo1 KO OCY454 cells at early (D1), intermediate (D14), and late (D28) stages of differentiation allowed for the identification of key signaling pathways in driving normal osteocyte dfiferentiation, as well as those regulated by Piezo1 mechanotransduction. To mechanistically investigate the role of Piezo1 in osteocyte differentiation, we generated a Piezo1 "knock-out" (KO) osteocyte cell line using CRISPR-Cas9 gene editing. Exon 19 through exon 21 from the OCY454 cell line were deleted, confirmed by sanger sequencing, qRT-PCR, western blot, and immunofluroescent staining. WT and Piezo1 KO OCY454 cells were differentiated for 1 (basal), 14, and 28 days at 37C and collected for bulk RNA-sequencing analysis. Two replicates were performed for each group.