Mechanical Stimulation Of Osteocyte-like Cells Changes Their Secretome - Implications For Regenerative Medicine

Abstract

Osteocytes are secretory bone cells that regulate bone homeostasis and for this reason, are often coined as the “brain of the bone”. In vitro studies demonstrated that mechanically stimulated osteocytes release interleukins and growth factors that help coordinating bone formation and resorption, however, their secretome remains largely unknown. PURPOSE: To investigate WNT signalling and the secretome of mouse and human osteocyte-like cells. Insights from this study could help to devise informed therapeutic exercise regimen e.g. aiming to preserve bone mass across ageing or accelerate bone fracture healing. METHODS: The murine MLO-Y4 (Kerafast) cell line was cultured according to Kerafast instructions. Human adipose stem cells (ATCC® PCS -500-011™) were expanded and differentiated into osteocyte-like cells (hOC) according to ATCC instructions. Cells were cultured in a computer-controlled bioreactor (Flexcell Int) for mechanical loading (3.4%, 2Hz, 5h). Static cultures were used as control. Relative expression of 84 key genes of the WNT signalling pathway (Sabiosciences) was quantified by RT-qPCR. Relative protein expression was estimated by western blotting. The secretome was analysed by quantitative mass spectrometry (TripleTOF 6600, SCIEX) using SWATH and IDA and processed using OneOmics (SCIEX) software. RESULTS: The relative gene expression remained unchanged in mechanically MLO-Y4 and hOC. Regarding protein quantification, active β-catenin and Cyclin D1 showed an up-regulation trend in mechanically stimulated MLO-Y4 but this was not statistically significant. A total of 917 proteins were identified in the MLO-Y4 secretome, ~12% present exclusively under mechanical active conditions. The secretome obtained under loading contained 14 cyclin-dependent kinases (CDKs) including CDK6, a critical regulator of osteoblasts and osteoclasts differentiation. A total of 329 proteins were identified in the supernatant of hOC, ~9% present exclusively under mechanical stimulation. Unlike MLO-Y4, no CDKs were identified in this cell type. The small ubiquitin-like modifier (SUMO) 2 and 3 were present in the secretomes of mechanically loaded MLO-Y4 and hOC. CONCLUSION: Mechanically stimulated osteocyte-like cells secrete a specific set of proteins which could impact bone health and regeneration.