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.
Highlights
Diseases related to osteoclast deficiency as well as osteoclast over-activity have been well described
The iRANK construct was transduced into a murine monocytic cell line, RAW264.7 and stably transduced cells were selected by FACS using GFP expression
A 70 kDa band consistent with the iRANK fusion protein was observed in the RAW264.7+iRANK cells but not in the nontransduced RAW264.7 cells by Western blot (Figure 1B)
Summary
Diseases related to osteoclast deficiency as well as osteoclast over-activity have been well described. Heterotopic ossification (HO) refers to abnormal deposition of calcium salts, often taking the form of bone in soft or hard tissues as a result of genetic mutation, trauma or disease [1]. Radiation therapy is effective when it is delivered to prevent HO, but it is not beneficial once HO is formed [5]. In HO, osteoblasts and mature bone are observed in calcified lesions, but a paucity of osteoclasts has been noted, consistent with a potential role of osteoclast deficiency in the etiology of this pathology. Osteoclasts have been proposed as a potential cell therapy to prevent or regress the mineral found in HO [7]
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