Abstract
Osteolytic bone disease is characterized by excessive osteoclast bone resorption leading to increased skeletal fragility and fracture risk. Multinucleated osteoclasts formed through the fusion of mononuclear precursors are the principle cell capable of bone resorption. Pregnenolone (Preg) is the grand precursor of most if not all steroid hormones and have been suggested to be a novel anti-osteoporotic agent. However, the effects of Preg on osteoclast biology and function has yet to be shown. Here we examined the effect of Preg on receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast formation and bone resorption in vitro, and potential therapeutic application in inflammatory bone destruction and bone loss in vivo. Our in vitro cellular assays demonstrated that Preg can inhibit the formation of TRAP+ve osteoclast formation as well as mature osteoclast bone resorption in a dose-dependent manner. The expression of osteoclast marker genes CTSK, TRAP, DC-STAMP, ATP6V0d2, and NFATc1 were markedly attenuated. Biochemical analyses of RANKL-induced signaling pathways showed that Preg inhibited the early activation of extracellular regulated protein kinases (ERK) mitogen-activated protein kinase (MAPK) and nuclear factor-κB, which consequently impaired the downstream induction of c-Fos and NFATc1. Using reactive oxygen species (ROS) detection assays, we found that Preg exhibits anti-oxidant properties inhibiting the generation of intracellular ROS following RANKL stimulation. Consistent with these in vitro results, we confirmed that Preg protected mice against local Lipopolysaccharide (LPS)-induced inflammatory bone destruction in vivo by suppressing osteoclast formation. Furthermore, we did not find any observable effect of Preg on osteoblastogenesis and mineralization in vitro. Finally Preg was administered to ovariectomy (OVX)-induced bone loss and demonstrated that Preg prevented systemic OVX-induced osteoporosis. Collectively, our observations provide strong evidence for the use of Preg as anti-osteoclastogenic and anti-resorptive agent for the potential treatment of osteolytic bone conditions.
Highlights
Osteolytic bone diseases such as inflammatory bone destruction and post-menopausal osteoporosis share a common theme of deterioration and destruction of the microstructure of bone tissue leading to marked decreases in bone mass, skeletal fragility, and increased risk of bone fractures
We showed that Preg can inhibit RANKLinduced osteoclastogenesis and bone resorption in a dosedependent manner in vitro and protected mice against LPSinduced bone destruction and ovariectomy-induced bone loss in vivo
We found that Preg treatment was associated with the inhibition of early RANKL-induced activation of ERK mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) signaling cascade which attenuated the induction of c-Fos and NFATc1
Summary
Osteolytic bone diseases such as inflammatory bone destruction and post-menopausal osteoporosis share a common theme of deterioration and destruction of the microstructure of bone tissue leading to marked decreases in bone mass, skeletal fragility, and increased risk of bone fractures. Osteoclasts are multinucleated giant cells formed through the fusion of mononuclear cells of the monocyte/macrophage cell lineage (Boyle et al, 2003). They are the principle and only cell in the body capable of resorbing and degrading the mineralized bone matrix (Lacey et al, 2012). RANKL on the other hand drives the differentiation and fusion of precursor cells toward the osteoclast lineage Both M-CSF and RANKL is required for subsequent mature osteoclast activation toward bone resorption (Udagawa et al, 1990; Lacey et al, 1998; Yasuda et al, 1998)
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