Targeting of the Osteoclastogenic RANKL–RANK Axis Prevents Osteoporotic Bone Loss and Soft Tissue Calcification in Coxsackievirus B3–Infected Mice

Abstract

Bone mineralization is a normal physiological process, whereas ectopic calcification of soft tissues is a pathological process that leads to irreversible tissue damage. We have established a coxsackievirus B3 (CVB3)–infected mouse model that manifests both osteoporosis and ectopic calcification specifically in heart, pancreas, and lung. The CVB3-infected mice showed increased serum concentrations of both cytokines including IL-1β, TNF-α, and the receptor activator of NF-κB ligand (RANKL) that stimulate osteoclast formation and of the osteoclast-derived protein tartrate-resistant acid phosphatase 5b. They exhibited more osteoclasts in bone, with no change in the number of osteoblasts, and a decrease in bone formation and the serum concentration of osteoblast-produced osteocalcin. These results indicate that CVB3-induced osteoporosis is likely due to upregulation of osteoclast formation and function, in addition to decreased osteoblast activity. In addition, the serum in the CVB3-infected mice contained a high inorganic phosphate content, which causes ectopic calcification. RANKL treatment induced an increase in the in vitro cardiac fibroblast calcification by inorganic phosphate via the upregulation of osteogenic BMP2, SPARC, Runx2, Fra-1, and NF-κB signaling. We finally observed that i.p. administration of RANK-Fc, a recombinant antagonist of RANKL, prevented bone loss as well as ectopic calcification in CVB3-infected mice. Thus, our results indicate that RANKL may contribute to both abnormal calcium deposition in soft tissues and calcium depletion in bone. In addition, our animal model should provide a tool for the development of new therapeutic agents for calcium disturbance in soft and hard tissues.

MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification

“…For detection of mineralization of RVSMCs, von Kossa staining was performed 53 . In brief, cells were fixed with 10% formalin for 30 min at room temperature, washed with dH 2 O three times and then incubated with 5% silver nitrate for 30 min at room temperature.…”

Silencing of SPARC represses heterotopic ossification via inhibition of the MAPK signaling pathway

“…SPARC, commonly known as an indicator of bone formation, also has the ability to promote calcium deposition as well as the differentiation and survival of osteoblasts [7]. Also, SPARC represents an osteogenic marker gene, and RANKL could promote Pi-induced cardiac fibroblast calcification in vitro by activating SPARC [8]. A previous study has since demonstrated that SPARC is highly positive in ligament fibers and the periosteal membrane attached to the apex of the styloid process [9].…”

Uremia Induces Dental Pulp Ossification but Reciprocally Inhibits Adjacent Alveolar Bone Osteogenesis

“…Meanwhile, the up-regulation of RANKL/RANK seems to resist uremic pulp ossification and further shift the imbalance toward osteogenesis. We also speculated that the influence of RANKL/RANK pathway might be different among different tissues, which was supported by a study showing that RANKL contributes to both calcium deposition in soft tissues but calcium depletion in bone [33].…”