Effects Of Osteoprotegerin On The Peripheral Adhesive Structures And Fusion Of (Pre)Osteoclasts

Defects in bone homeostasis are a major health problem. Osteoclast differentiation and activation have a crucial role in bone remodeling in health and disease. Osteoclasts are bone-resorbing cells and are formed through cell fusion of mononuclear macrophage/monocyte-lineage hematopoietic precursors. Osteoclasts attach to the extracellular matrix via specialized attachment structures called podosomes, which form a prominent F-actin-rich ring that is thought to correspond to the sealing zone of resorbing osteoclasts. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB (RANK) are members of the tumor necrosis factor receptor (TNFR) superfamily that regulate osteoclast formation and function by competing for RANK ligand (RANKL). RANKL promotes osteoclast development through RANK activation, while OPG inhibits this process by sequestering RANKL. Several studies have provided important details about the effects of OPG on the differentiation, fusion and adhesion of osteoclasts. This research has mainly been focused on the expression of genes encoding osteoclast specific markers, potential fusion factors, and podosome related modulators, in vitro blocking, or overexpression. As a result, many details in the molecule mechanisms of OPG-induced inhibitory effects on osteoclast differentiation, fusion and adhesion remain unclear. In this study, RAW264.7 murine monocytic cells treated with M-CSF and RANKL were used to produce osteoclasts. Then, various concontrations of OPG were added into the culture system. Morphological observation, Real time cell analysis (RTCA) and Western blot were performed to survey whether OPG disrupts podosome and inhibits fusion of osteoclasts or precursors and characterize the mechanisms underlying the influence of OPG on osteoclasts or precursors. A series of tests were carried out:1. Effects of OPG on the formation and function of osteoclasts at different stages of differentiationThis study aimed to investigate the effects of OPG on differentiation, adhesion and activity of osteoclasts from the various stages of differentiation. RAW264.7 cells were incubated with M-CSF and RANKL for 1,3,5, or 7 d, followed by an additional 24-h incubation in the presence or absence of OPG (80 ng/mL). We examined osteoclast differentiation and adhesion capacity using the tartrate-resistant acid phosphatase (TRAP) assay and immunofluorescence microscopy, and additionally examined cell growth in real time using the xCELLigence system. Furthermore, the expression levels of TRAP, RANK, integrin β3, matrix metalloproteinase 9 (MMP9), cathepsin K, carbonic anhydrase Ⅱ (CA Ⅱ), and vesicular-type H+-ATPase Al (V-ATPase A1) were examined using western blotting. OPG exposure on day 1 enhanced the osteoclast growth curve and as well as adhesion, and increased RANK and integrin (β3 expression. In contrast, exposure to OPG at later time points (days 3 to 7) inhibited osteoclast differentiation, adhesion structure formation, and protease expression. In conclusion, the biological effects of OPG exposure at the various stages of osteoclast differentiation were varied, and included the enhanced adhesion and survival of preosteoclasts, the block of differentiation from the early to the terminal stages of osteoclastogenesis, and suppression of mature osteoclast activation following OPG exposure during the terminal differentiation stage, suggesting that the effects of OPG exposure differ based on the stage of differentiation.2. Mechanism involved in the effects of OPG on peripheral adhesive structures of osteoclastsTo study the detailed mechanisms of OPG-induced disassembly of podosomes, disruption of adhesive structures and modulation of adhesion-related proteins in osteoclasts, RAW264.7 cells were incubated with M-CSF and RANKL for 3 d, followed by an additional 24-h incubation in the presence or absence of different concentrations of OPG (0,20,40 and 80 ng/mL). TRAP staining demonstrated that OPG inhibited differentiation of osteoclasts. The use of scanning electron microscopy (SEM), RTCA and confocal microscopy indicated that osteoclasts responded in a time and dose-dependent manner to OPG treatments with retraction of peripheral adhesive structures and detachment from the extracellular substrate. Combined imaging and Western blot studies showed that OPG induced dephosphorylation of Tyr 402 in Pyk2 and decreased its labeling in peripheral adhesion regions. OPG induced increased intracellular labeling of Tyr 402 in Pyk2, Tyr 416 in Src, increased dephosphorylation of Tyr 527 in Src, and increased Pyk2/Src association in the central region of osteoclasts. This evidence suggests that Src may function as an adaptor protein that competes for Pyk2 and relocates it from the peripheral adhesive zone to the central region of osteoclasts in response to OPG treatment. OPG may induce podosome reassembly and peripheral adhesive structure detachment by modulating phosphorylation of Pyk2 and Src and their intracellular distribution in osteoclasts.3. Calcium, ERK, and p38 MAPK signaling in OPG-induced podosome disassembly of osteoclastsTo characterize the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts, this study 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 Ca2+ concentration [Ca]i and the phosphorylation state of podosome associated proteins (Pyk2 and Src). RAW 264.7 cells were differentiated to osteoclasts using RANKL and M-CSF. The cells were pretreated with Bapta-AM (5 μM), U0126 (5 μM), or SB202190 (10 μM) for 30 minutes, followed by 40 ng/mL OPG for 3 hours. Osteoclastogenesis, adhesion structures, viability and morphology, [Ca2+]i and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, SEM, RTCA, 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 [Ca2+]i, 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.4. Mechanisms involved in OPG-induced the inhibition of fusion between (pre)osteoclastsTo research the detailed mechanisms of OPG-induced inhibited fusion of (pre)osteoclasts, this study investigated the effects of ATP, OPG or the combination of both on (pre)osteoclast differentiation, fusion and important factors involving fusion. RAW 264.7 cells were cultured with RANKL and M-CSF for 3 d to generate osteoclasts, then treated with 100 μM ATP/40 ng/mL OPG/combination of ATP and OPG for additional 2 d. Differentiation, fusion rates, the expression levels and localization of CD44, CD47, DC-STAMP, ATP6V0D2, or Connexin43, factors involving (pre)osteoclast fusion, in this culture system were measured by TRAP staining, RTCA, western blotting, and confocal microscopy respectively. OPG significantly inhibited the formation of multinucleated osteoclasts, while ATP partially restored osteoclastogenesis. For preosteoclasts, OPG only reduced the amount of CD47, but decreased the expression of CD44, CD47, DC-STAMP, ATP6V0D2 and total Connexin43 derived from multinucleated osteoclasts. ATP restored the amount of all of them. Imaging study showed that OPG induced CD44-, CD47-DC-STAMP-, ATP6V0D2- or Connexin43-positive (pre)osteoclasts declined. These results suggest OPG may inhibit the fusion process of preosteoclasts and mature osteoclasts by different mechanisms and deprive (pre)osteoclasts of the production of ATP.