Search

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]

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]