title: An inducible, ligand-independent receptor activator of NF-κB gene to control osteoclast differentiation from monocytic precursors.
abstract: Osteoclasts are bone-resorbing cells that are critical for the normal formation and maintenance of teeth and skeleton. Osteoclast deficiency can contribute to heterotopic ossification (HO), a pathology that is particularly detrimental to the mechanical functions of joints, valves and blood vessels. On the other hand, osteoclast over-activity is a major cause of osteoporosis. A reliable method for controlled generation of osteoclasts would be useful as a potential autologous cell therapy for HO, as well as high-throughput drug screening for anti-osteoporotic drugs. In this report, we describe the development of a cell engineering approach to control monocytic precursor cell differentiation to osteoclasts. Oligomerization of receptor activator of nuclear factor κB (RANK) is known to be essential for osteoclast differentiation from monocyte/macrophage precursors. We engineered a murine monocytic cell line, RAW264.7 to express a fusion protein comprising the intracellular RANK signaling domain and FK506-derived dimerization domains that bind to a small molecule chemical inducer of dimerization (CID). Virally infected cells expressing this fusion protein were treated with CID and dose-dependent induction of tartrate-resistant acid phosphatase activity, as well as multinucleated osteoclast formation were observed. Furthermore, NF-κB signaling was upregulated in a CID-dependent fashion, demonstrating effective RANK intracellular signaling. Functionally CID-induced osteoclasts had robust mineral resorptive activity in both two-dimensional and three-dimensional in vitro resorption assays. In addition, the CID-induced osteoclasts have the same life span as native RANKL-induced osteoclasts. Most importantly and crucially, the engineered cells differentiated into osteoclasts that were resistant to the potent osteoclast inhibitor, osteoprotegerin. Taken together, these studies are the first to describe a method for inducible control of monocytic precursor differentiation to osteoclasts that may be useful for future development of an engineered autologous cell therapy as well as high-throughput drug testing systems to treat diseases of osteoclast over-activity that are independent of osteoprotegerin.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Cameron W
authors.initials: CW
authors.lastname: Rementer
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Meiting
authors.initials: M
authors.lastname: Wu
authors.affiliation: Department of Oral Health Sciences, University of Washington, Seattle, Washington, United States of America.
authors.forename: Worakanya
authors.initials: W
authors.lastname: Buranaphatthana
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Hsueh-Ying L
authors.initials: HY
authors.lastname: Yang
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Marta
authors.initials: M
authors.lastname: Scatena
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Giachelli
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D023461:Myeloid Progenitor Cells / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*
chemical_list: D016328:NF-kappa B; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse