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.

Osteoclast precursors do not express CD68: results from CD68 promoter‐driven RANK transgenic mice

“…We previously validated the same iRANK construct using a murine preosteoclastic cell line, RAW264.7. When treated with 50 n m of CID, iRANK‐expressing RAW264.7 cells showed induction of tartrate‐resistant acid phosphatase (TRAP) activity as well as formation of mineral resorbing multinucleated osteoclasts . The current studies confirmed the expression and function of CD68‐driven iRANK in macrophage precursors of transgenic mice, but no expression or function of this construct was observed in bone marrow‐derived (BMD) or spleen‐derived osteoclast precursors.…”

“…The iRANK dimerization domain allows interaction with a chemical inducer of dimerization (CID) drug. As RANK requires trimerization for effective signaling, we have engineered two FK506‐derived dimerization domains fused to the RANK cytoplasmic domain to ensure successful oligomerization . Thus, in this system, the fusion protein (iRANK) is functionally inert unless directed to oligomerize by the addition of the CID, AP20187.…”

Engineering macrophages to control the inflammatory response and angiogenesis

“…Further, our lab has previously demonstrated that this system can be used to engineer inducible bone resorbing osteoclasts from the monocyte-macrophage RAW264.7 cell line, in which it is important to note that monocytes are a common precursor to both macrophages and osteoclasts. [27]…”

Calcific Aortic Valve Disease

“…Tissue targeting may be essential to avoid damage to skeletal bone; for example, if osteoclasts or nanoparticles carrying osteoclastic differentiation factors could be targeted to valve tissue, they may reverse CAVD. [6] However, even if successful, mineral resorption may leave behind damaged leaflets. Molecular imaging of these events may provide biomarkers for clinical and research monitoring far in advance of critical stenosis.…”

“…[49] Furthermore, new techniques to engineer monocytes that conditionally differentiate into OPG-resistant osteoclasts have been recently developed. [6] Thus, approaches to control osteoclast differentiation and/or locally deliver autologous osteoclasts represent potential novel therapeutic modalities to treat or even regress CAVD.…”