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Objective To investigate the effect of boldine isolated from Litsea cubeba on collagen-induced arthritis (CIA) rats and explore the molecular mechanism predicted by network pharmacology. Material and methods CIA rats were orally administered with boldine. The bone destruction of paws was analyzed by histologic examination, tartrate-resistant acid phosphatase (TRACP) staining and micro-computed tomography. Prediction of signal pathway associated with boldine network molecules and CIA genes was applied by the network pharmacology analysis. The expressions of osteoprotegerin (OPG), receptor activator of nuclear factor-κB (RANK) and its ligand (RANKL) in the ankle were detected by immunohistochemistry. In vitro osteoclasts were cultured in the presence of variable doses of boldine and the RANK expressions were evaluated using Real-time polymerase chain reaction and western blot. Results Boldine reduced ankle swelling, alleviated pathological damage and significantly prevented bone destruction in CIA rats. Consistent with this, enzyme linked immunosorbent assay revealed boldine decreased serum TRACP5b levels and osteoclast number in the ankle region by TRACP staining from CIA rats. The network pharmacology analysis indicated that RANK signaling in osteoclasts was the most significant canonical pathway associated with boldine network molecules and CIA genes, which was verified by the increased expression of OPG, reduced expression of RANK, RANKL and RANKL/OPG in boldine-treated CIA rats. The in vitro study further confirmed that boldine inhibited osteoclastogenesis by inhibiting the RANKL/RANK signaling pathway. Conclusion Taken together, our study first indicates that boldine from Litsea cubeba suppresses osteoclastogenesis, improves bone destruction by down-regulating the OPG/RANKL/RANK signal pathway and may be a potential therapeutic agent for rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca 2+ concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30 ng/mL) and M-CSF (25 ng/mL). The cells were pretreated with Bapta-AM (5 μM), U0126 (5 μM), or SB202190 (10 μM) for 30 min, followed by 40 ng/mL OPG for 3 h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca 2+ concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca 2+ concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix. [ABSTRACT FROM AUTHOR]

Bone is continuously remodeled in a dynamic process maintained by osteoclasts and osteoblasts, and imbalances in the relative activities of these cell types can cause various pathological conditions, including rheumatoid arthritis and osteoporosis. Osteoclasts are multinucleated cells that serve an important role in regulating the development of osteoporosis. Furthermore, P2X7 receptor activation has a vital role in physiological and pathological reactions in bone, including bone disease. Therefore, the present study aimed to investigate the effect of P2X7 receptor on osteoclast differentiation and to explore the underlying molecular mechanism by western blotting and tartrate-resistant acid phosphatase staining. The results indicated that the expression levels of P2X7 receptor and intracellular Ca2+ concentration levels were very high in mature osteoclasts. Furthermore, P2X7 receptor overexpression increased the number of multinucleated osteoclasts and the expression of osteoclastogenesis-related proteins. P2X7 receptor overexpression was also associated with downstream activation of Ca2+/calcineurin/nuclear factor of activated T cells c1 (NFATc1) signaling and increased expression of autophagy-related proteins during osteoclast differentiation. By contrast, knockdown of P2X7 receptor exerted the opposite effects. Notably, FK506 (a Ca2+/calcineurin/NFATc1 signaling inhibitor) abrogated P2X7 receptor overexpression-induced osteoclast differentiation and activation of autophagy. Moreover, 3-MA (an autophagy inhibitor) significantly suppressed P2X7 receptor overexpression-induced osteoclast differentiation. In conclusion, P2X7 receptor knockdown may suppress osteoclast differentiation by modulating autophagy and the Ca2+/calcineurin/NFATc1 signaling pathway. [ABSTRACT FROM AUTHOR]

Schematic representation of the proposed Cd-induced signaling pathway in osteoclasts. Treatment of osteoclasts with Cd leads to a transient increase in intracellular calcium concentration ([Ca2+] i). Cd-induces the elevation of [Ca2+] i and activation of CaM/CaMKII/CaMKIV signaling. CaM/CaMKII/CaMKIV signaling induces the expression of NFATc1, leading to osteoclast differentiation. ER Ca2+ release, the elevation of [Ca2+] I , and the activation of CaM, CaMKII, and CaMKIV can be inhibited by 2-APB, BAPTA-AM, W-7, KN93 and STO-609, respectively. Environmental cadmium (Cd) pollution can ultimately lead to chronic toxicity via food consumption. Previous studies have demonstrated that long-term low-dose Cd exposure decreases bone mineral density and bone mineralization. Cd may increase receptor activator of nuclear factor-κ B ligand (RANKL) expression by osteoclasts, and inhibit the expression of osteoprotegerin. However, the molecular mechanism underlying Cd toxicity toward osteoclasts is unclear. In this study, bone marrow monocytes were isolated from C57BL/6 mice and treated with macrophage colony-stimulating factor and RANKL to induce the formation of osteoclasts. The results show that low-dose Cd exposure induced osteoclast differentiation. Cd also increased the intracellular calcium concentration of osteoclasts by triggering release of calcium ions from the endoplasmic reticulum into the cytoplasm. Furthermore, the elevation of intracellular calcium levels was shown to activate the calmodulin (CaM)/calmodulin-dependent protein kinase (CaMK) pathway. NFATc1 is a downstream protein of CaM/CaMK signaling, as well as a key player in osteoclast differentiation. Overall, we conclude that Cd activates the CaM/CaMK/NFATc1 pathway and regulates osteoclast differentiation by increasing intracellular calcium concentration. Our data provide new insights into the mechanisms underlying osteoclast differentiation following Cd exposure. This study provides a theoretical basis for future investigations into the therapeutic application of CaMK inhibitors in osteoporosis induced by Cd exposure. [ABSTRACT FROM AUTHOR]