title: Identification of RANKL-Regulated Genes Involved in Osteoclast Differentiation and Resorption
Creators: name: Selinger, Christina Imanta
Publisher: name: Griffith University
Year: 2008
Subjects: subject: Peripheral blood mononuclear cells
subject: BPMCs
subject: Osteoclast differentiation
subject: siRNA
subject: RANKL

description: Peripheral blood mononuclear cells (PBMCs) are pluripotent for osteoclast and macrophage cell lineages. The differentiation of macrophages and osteoclasts from a common monocyte precursor is induced following exposure to macrophage-colony stimulating factor (M-CSF), or both M-CSF and receptor activator of nuclear factor B ligand (RANKL) respectively. Differential gene expression resulting from cytokine treatment of PBMCs was examined over time using differential display PCR (DD-PCR) and quantitative real-time PCR (Q-PCR). Q-PCR analysis verified the expression of a new chemokine, FAM19A1, in addition to Special AT-rich binding sequence 1 (SATB1), solute carrier family 16 member 6 (SLC16A6) and LIM kinase 1 (LIMK1) in primary human osteoclasts, however, only LIMK1 was significantly up-regulated by RANKL. Highly efficient delivery of small interfering RNA (siRNA) transfection to primary human osteoclasts was developed, and represents a technical milestone due to the inherent phagocytic tendencies of the PBMC lineage. The development of RNA interference for use in primary human osteoclasts was conducted using siRNA synthesised by Dicer enzyme, to verify the role of candidate genes in osteoclast differentiation and osteoclast bone resorption. Cathepsin K (CTSK) is the key proteinase expressed by osteoclasts, and was used as a benchmark for the optimisation of siRNA inhibition in primary human osteoclasts. Transfection of primary human osteoclasts with siRNA to CTSK significantly diminished bone resorption, with a 60% reduction in area resorbed (P=1.3x10-2), and a 50% reduction in pit number (P=1.8x10-2). Normal bone remodelling is dependent on both the rate of osteoclast formation and resorption. A number of genes were examined for their contribution to osteoclast formation and resorption using siRNA. Nuclear factor of activated T cells, calcineurin dependant 1 (NFATc1) inhibition was found to significantly deplete osteoclast formation (P=4.0x10-3), confirming other NFATc1 inhibition studies, and the necessity of NFATc1 in osteoclast differentiation. In pre-differentiated osteoclasts, siRNA targeting NFATc1 did not reduce osteoclast bone resorption, rather it significantly increased area resorbed (P=1.0x10-3), with no significant difference in cell number. This result suggests that NFATc1 may act in accordance with its regulator calcineurin, which has been found to enhance osteoclast differentiation, but inhibit osteoclast resorption in mature cells. The inhibition of LIMK1 by targeted small interferring RNA (siRNA) was found to significantly diminish osteoclast formation (P=1.0x10-3), pits resorbed (P=4.2x10-2), as well as area resorbed (P=4.0x10-3). LIMK1 is a signalling kinase, identified as RANKL-regulated in murine osteoclasts, notwithstanding, this is the first study that confirms LIMK1 involvement in osteoclast formation and activity. LIMK1/cofilin-mediated actin reorganisation is critical to progenitor cell migration to stromal cells, and also regulates the stability of F-actin formation. F-actin rings were analysed in LIMK1 depleted pre-differentiated osteoclasts, which seemed to have formed properly and did not appear dissimilar from controls.