Stage-Specific Modulation of Multinucleation, Fusion and Resorption by the Long Non-coding RNA DLEU1 and miR-16 in Human Primary Osteoclasts

Abstract

Osteoclasts are multinucleated cells formed through fusion of mononucleated precursors of the myeloid lineage and are the only cells that can resorb all the constituents of the bone matrix. Our goal was to investigate the role of long non-coding RNA DLEU1 and miR-16-5p in the fusion of human primary osteoclasts and their resorptive capacity. We found DLEU1 to be markedly upregulated, whereas miR-16 was significantly suppressed, during osteoclast differentiation, suggesting a potential involvement in the multinucleation process. The knockdown of DLEU1 or the overexpression of miR-16 in human primary pre-osteoclasts from male human donors (50 years or older) impaired fusion at both early and late time-points, each in distinct ways, without affecting cell viability. Time-lapse recordings confirmed the impairment of the fusion process and showed an abrogation of the phagocytic cup fusion modality, as well as a reduction of the fusion between mononucleated precursors and multinucleated osteoclasts during DLEU1 silencing. Furthermore, mass spectrometry-based quantitative proteomics revealed that the effects of DLEU1 and miR-16 on osteoclast fusion were mediated by distinct proteins and processes. Thus, both DLEU1 inhibition and/or miR-16 overexpression hinder osteoclast fusion through modulation of different mechanisms. Moreover, decreased levels of DLEU1 specifically affected the resorption speed of pit-making osteoclasts, while increased levels of miR-16 promoted bone resorption mainly through pit-formation, impairing the resorption speed of the osteoclasts making trenches and affecting their resorbed area. Taken together, these findings identify DLEU1 and miR-16 as mediators of osteoclast fusion and activity, offering potential new therapeutic targets to ameliorate bone destruction in skeletal diseases with accentuated bone deterioration.