Silencing Itch in human peripheral blood monocytes promotes their differentiation into osteoclasts

Abstract

ntroduction Two clinical case reports of humans with mutations in Itch reported distinct morphological defects such as stunted growth, macrocephaly, and dysmorphic features indicating a role for Itch in bone remodelling. Studies in mice have found that the encoded E3 ubiquitin ligase acts as a negative regulator of osteoclastogenesis, however no studies have investigated whether this is translatable to a human model. Experimental procedures Human peripheral blood monocytes were separated from whole blood and grown in M-CSF containing media. Media was later supplemented with RANKL to promote osteoclast differentiation. Transient siRNA-mediated Itch knockdown (si-Itch) in monocytes was verified by qPCR and western blot to confirm reduction in both Itch mRNA and protein respectively. Monocytes were aliquoted onto 96-well plates where confluence and osteoclast formation were analysed using automated cytometry analysis before and after staining for tartrate resistant acid phosphatase activity (TRAP). Cells were also stained with Hoechst33342 to look for multinucleate cells. Results Cells treated with si-Itch showed an 80% knockdown in Itch mRNA and > 75% reduction in protein. Following the 7-day differentiation period, si-Itch caused a 47% increase in multinucleate cells and a 17% increase in numbers of large cellular bodies and, indicating an overall increase in mature osteoclast formation. Conclusions Our preliminary data shows silencing Itch expression increases the potential of primary human monocytes to differentiate into osteoclast-like cells in vitro.