Abstract
Rheumatoid arthritis is an autoimmune disease characterized by synovial hyperplasia and progressive joint destruction. As reported previously, recombinant human endostatin (rhEndostatin) is associated with inhibition of joint bone destruction present in rat adjuvant-induced arthritis; however, the effect of rhEndostatin on bone destruction is not known. This study was designed to assess the inhibitory effect and mechanisms of rhEndostatin on formation and function of osteoclasts in vitro, and to gain insight into the mechanism underlying the inhibitory effect of bone destruction. Bone marrow-derived macrophages isolated from BALB/c mice were stimulated with receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor to establish osteoclast formation. Osteoclast formation was determined by TRAP staining. Cell viability of BMMs affected by rhEndostatin was determined using a MTT assay. Bone resorption was examined with a bone resorption pits assay. The expression of osteoclast-specific markers was analyzed using quantitative real-time PCR. The related signaling pathways were examined using a Luciferase reporter assay and western blot analysis. Indeed, rhEndostatin showed a significant reduction in the number of osteoclast-like cells and early-stage bone resorption. Moreover, molecular analysis demonstrated that rhEndostatin attenuated RANKL-induced NF-κB signaling by inhibiting the phosphorylation of IκBα and NF-κB p65 nuclear translocation. Furthermore, rhEndostatin significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases, such as ERK1/2, JNK, and p38. Hence, we demonstrated for the first time that preventing the formation and function of osteoclasts is an important anti-bone destruction mechanism of rhEndostatin, which might be useful in the prevention and treatment of bone destruction in RA.
Highlights
Severe destruction of the adjacent cartilage and bone is the pathologic hallmark of rheumatoid arthritis (RA), and affects one’s ability to do physical activities, thereby reducing the quality of life (Ochi et al, 2007; Wei et al, 2013)
The cytosolic level of phosphorylation of IκBα was detected by western blot, and we showed that RANKL induced the cytosolic level of phosphorylation of IκBα reached the maximum level after 5 min, whereas the cytosolic level of phosphorylation of IκBα was markedly decreased by pre-treatment with rhEndostatin in bone marrow monocytes (BMMs) (Figures 5B,C)
Our results suggested that rhEndostatin suppresses osteoclastogenesis by attenuating the activation of mitogen-activated protein kinase (MAPK) pathways induced by RANKL
Summary
Severe destruction of the adjacent cartilage and bone is the pathologic hallmark of rheumatoid arthritis (RA), and affects one’s ability to do physical activities, thereby reducing the quality of life (Ochi et al, 2007; Wei et al, 2013). It has been shown that osteoclasts play a critical role in local bone erosion in RA joints (Gravallese, 2002). In RA, numerous multinucleated rhEndostatin Inhibits Osteoclast Formation osteoclast-like cells are present at sites of bone erosion, followed by degradation of the bone matrix (Pettit et al, 2006). It has been demonstrated that bone erosion does not appear in osteoclast-deficient mice in arthritis models (Pettit et al, 2001). High receptor activator of NF-κB ligand (RANKL), an essential factor for osteoclast formation, has been detected in the synovium of RA patients (Lubberts et al, 2002).
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