Recombinant human endostatin inhibits TNF-alpha-induced receptor activator of NF-kB ligand expression in fibroblast-like synoviocytes in mice with adjuvant arthritis

Bone loss is a critical pathology responsible for the functional disability in patients with rheumatoid arthritis (RA). It is well known that receptor activator of nuclear factor kappa-B (NF-kB) ligand (RANKL) plays a crucial role in bone loss in RA. The purpose of this study was to determine whether recombinant human endostatin (rh-endostatin) mediates bone erosion in RA by regulation of RANKL expression in an experimental model of RA, consisting of mice with adjuvant-induced arthritis (AA). Cultured AA fibroblast-like synoviocytes (FLSs) obtained from these mice were induced by tumor necrosis factor-alpha (TNF-alpha) combined with or without rh-endostatin. The levels of RANKL and osteoprotegerin (OPG) mRNA, soluble and membrane-bound proteins were assessed by real-time PCR, ELISA, and Western blotting. Western blotting and the luciferase reporter assay were used to study related signaling pathways. Rh-endostatin inhibited RANKL mRNA expression, soluble and membrane-bound protein expression in AA FLSs but not in CD4+T cells. However, OPG expression and secretion was not affected by rh-endostatin in AA FLSs. Molecular analysis demonstrated that rh-endostatin significantly inhibited TNF-ainduced MAPK and AP-1 signaling pathways. Moreover, rh-endostatin attenuated TNF-alpha-induced NF-kB signaling by suppressing the phosphorylation level of inhibitor kappaBa (IkB alpha) and nuclear translocation of NF-kB p65 in FLSs from mice with AA. These results provide the first evidence that rh-endostatin inhibits TNF-alpha-induced RANKL expression in AA FLSs.