Activation of RANK by its ligand leads to theexpression of osteoclast-specific genes during differentiation, andthe activation of resorption by mature osteoclasts (). RANK signaling is mediated bycytoplasmic factors that activate downstream pathways controllingthese various functions. At least five distinct proteinkinase-mediated signaling cascades are induced duringosteoclastogenesis and activation; inhibitor of NF-κB kinase (IKK),c-Jun N-terminal kinase, p38, ERK and Src pathways (). ERK is essential for osteoclastdifferentiation, survival and activation (,,,). The inhibition of ERK has beenproven to possess a therapeutic potential for osteoclast-relateddiseases (). Activated ERKstimulates transcription factors, such as NFATc1 (), which is a master regulator ofosteoclast differentiation (,,). Overexpression of NFATc1accelerates RANKL-induced osteoclast differentiation and can alsoincrease osteoclast formation independently of RANKL. Additionally,NFATc1-deficient embryonic stem cells failed to differentiate intoosteoclasts, even in the presence of RANKL (). Using western blot analysis, AS-IVwas revealed to inhibit RANKL-induced ERK signaling. NF-κB wasunaffected and this result was confirmed using an NF-κB luciferasereporter assay. AS-IV also inhibited NFATc1 mRNA and proteinexpression. NFATc1 regulates the expression of a range of genesassociated with osteoclast differentiation and function. In thepresent study, the expression of NFATc1-regulated genes (TRAP, CtsKand V-ATPase d2) was downregulated by AS-IV, suggesting that AS-IVnot only affected the expression of NFATc1, but also affected theexpression of its downstream genes.
A high-resolution micro-CT scanner (Skyscan 1176;Skyscan; Aartselaar, Belgium) was used with the following settings:X-ray voltage, 50 kV; electric current, 500 mA; and rotation step,0.7°. Following reconstruction, a square region of interest (ROI)around the midline suture was chosen for further qualitative andquantitative analyses. The bone volume against tissue volume(BV/TV), number of porosities and percentage of total porosity weredetermined for each sample as described previously ().
, the skeleton is constantly beingremodeled via a process involving the breakdown (resorption) andbuild-up (synthesis) of bone, determined by a delicate balancebetween osteoblast and osteoclast activities (). As osteoclasts have key roles in theregulation of bone mass and quality, the majority of adult skeletaldiseases are due to excess osteoclast activity, resulting inosteopenia (,). Such diseases include osteoporosis,periodontal disease, rheumatoid arthritis and aseptic prosthesisloosening (,). For individuals with osteoporosis, acondition characterized by low bone mass and skeletal fragility,low trauma bone fractures represent life-threatening events,particularly when they affect the vertebrae, proximal femur (hip),distal forearm or proximal humerus ().