SiRNA Knock-Down of RANK Signaling to Control Osteoclast-Mediated Bone Resorption

Abstract

To demonstrate the ability of small interfering (si)RNA targeting the cell receptor, RANK, to control osteoclast function in cultures of both primary and secondary osteoclasts and their precursor cells. siRNA targeting RANK was transfected into both RAW264.7 and primary bone marrow cell cultures. RANK knock-down by siRNA and functional inhibition were assessed in both mature osteoclast and their precursor cell cultures. RANK mRNA message and protein expression after the transfections were analyzed by PCR and Western blot, respectively. Off-target effects were assessed. The inhibition of osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) assay, and subsequent bone resorption was determined by resorption pit assay. Both osteoclasts and osteoclast precursors can be targeted by siRNA in serum-containing media. Delivery of siRNA targeting RANK to both RAW 264.7 and primary bone marrow cell cultures produces short term repression of RANK expression without off-targeting effects, and significantly inhibits both osteoclast formation and bone resorption. Moreover, data support successful RANK knock-down by siRNA specifically in mature osteoclast cultures. RANK is demonstrated to be an attractive target for siRNA control of osteoclast activity, with utility for development of new therapeutics for low bone mass pathologies or osteoporosis.