Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling

Abstract

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 μM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 μM), and E-64 (IC50 3 μM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14(+) human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK.

Up‐regulated CST5 inhibits bone resorption and activation of osteoclasts in rat models of osteoporosis via suppression of the NF‐κB pathway

“…RANKL has been revealed to be associated with osteoporosis in postmenopausal women . An experiment had stated that RANKL can stimulate osteoclast formation . Another study has illustrated that osteoclastogenesis, bone resorption and osteoclast activities are inhibited through suppression of RANKL pathway .…”

“…32,33 An experiment had stated that RANKL can stimulate osteoclast formation. 34 Another study has illustrated that osteoclastogenesis, bone resorption and osteoclast activities are inhibited through suppression of RANKL pathway. 35 RANK is located in the cell membrane of osteoclasts and facilitates osteoclast activation and differentiation.…”

CCL11, a novel mediator of inflammatory bone resorption

“…After 5 and 6 days, all bone slices were TRAP-stained the number of TRAP positive cells with three or more nuclei were counted as osteoclasts and the size and area of resorption pits was measured in reflected light 36 .…”

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells, and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

“…Peripheral blood mononuclear cells were purified from buffy coats donated by five healthy males who had given their informed consent (Karolinska University Hospital Laboratory, Huddinge, Sweden) using density gradient centrifugation on Ficoll Paque Plus (GE Healthcare, Uppsala, Sweden). For more uniform differentiation of osteoclasts, CD14‐positive fraction of PBMCs was selected with CD14 + magnetic microbeads, according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD14 + PBMCs were cultured in 96‐well plates (NUNC, delta surface) in α‐MEM, supplemented with 10% FBS, GlutaMAX (2 mmol/L), streptomycin (100 μg/mL), penicillin (100 U/mL) (all from GIBCO, Grand Island, NY, USA), either on plastic surface (150 000 cells/well) or on 130 μm thick bovine cortical bone slices (250 000 cells/well) and differentiated into multinuclear cells for 6‐12 days in the presence of recombinant human M‐CSF (30 ng/mL; R&D Systems, Minneapolis, MN, USA) and sRANKL (2 ng/mL; Peprotech, Rocky Hill, NJ, USA) with or without aminothiazoles TH‐848 (0.2 μmol/L) or TH‐644 (2 or 15 μmol/L) (Chem‐Bridge Corp.).…”