| Osteoclasts have unique bone resorption activity. The dynamic equilibrium between osteoclast-mediated bone resorption and osteogenesis plays an important role in the bone remodeling process. Excessive bone resorption can cause a variety of diseases, such as osteoporosis, rheumatoid arthritis, multiple myeloma and periodontitis. Therefore, the study on osteoclast differentiation and bone absorption by osteoclasts is crucial for the development of effective therapeutic strategies.In the physiological or pathological condition of the organism, the recetor activator of nuclear factor-κB ligand, RANKL combined with the recetor activator of nuclear factor-KB, RANK to promote the differentiation and maturation of osteoclasts. Osteoprotegerin (OPG) are secreted by osteoblasts. OPG is the decoy receptor of RANKL and competes with RANK for RANKL, with antagonist effect for RANK. It is through this mechanism that the osteoclast differentiation and bone absorption are inhibited.Cytoskeleton is the network structure in the cytoplasm of eukaryotic cells, playing a significant role in some aspects like the morphology of the cells and the cells migration. Cytoskeleton is also involved in the transportation of materials in cells, information transference, energy transference, cells differentiation and division. Researches showed that the activity of bone resorption in osteoclasts has a bearing on cytoskeleton. Studies of the effect of OPG on bone resorption by osteoclasts have focused mainly on the OPG-induced inhibition of osteoclast differentiation via the OPG/RANKLRANK system. Though cytoskeleton played an important role in the process of differentiation and bone resorption of osteoclasts, it is few reported that the changes and molecular mechanisms of cytoskeleton together with the influence OPG has on cytoskeleton. This experiment is aimed to do research in the alteration of cytoskeleton and the regulatory mechanisms of Rho GTPases family and the impact OPG on osteoclast during differentiation and bone resorption of osteoclasts.1. Purification and culture of osteoclastOsteoclasts are terminal cells, which can not have division growth and the amount of osteoclasts is rare. In order to acquire massive active osteoclasts, we did some research on using RANKLE and M-CSF inducing RAW264.7 cells to differentiating to osteoclasts.We inoculated 4×103,8×103,1.6×104 cell/mL RAW264.7 cells to the 6 well plates, disposing cells with 60 ng/mL RANK and 50 ng/mL M-CSF. The study found that, cells of which the dense is 1.6 x 104 cell/mL were cultivated for 2 days and then a large amount of monocytes went to apoptosis along with creating osteoclasts, so did cells with dense of 8×103 cell/mL, cultivating for 3 days. However, after cultivating cells of 4×103cell/mL for 4 days, massive monocytes went to apoptosis along with creating osteoclasts.Comparing between the different quantity and volume of osteoclasts with different noculum density, we found that 4 x 103 cell/mL osteoclasts had the largest quantity and volume, which demonstrated that low noculum density together with longtime induction was beneficial to the differentiation of osteoclasts.2. Changes of cytoskeleton in process of osteoclast differentiationThe morphology and cytoskeleton during osteoclast differentiation from RAW264.7 were detected by using RTCA, TRAP staining, immunofluorescence staining. Moreover, the expressions of Rho GTPases along with the enzymes related to osteoclast differentiation and bone resorption were detected by QRT-PCR. As the results showed, the differentiation and maturation process of OC was divided into four stages, during which intensive changes occurred to cytoskeleton, cytomorphology and relevant regulatory factors:(1) Induced differentiation of RAW264.7 cells into osteoclast precursors, i.e. day 0-1; (2) Incubation period of osteoclast precursors, i.e. day 1-3; (3) transformation of osteoclast precursors into immature osteoclasts, i.e. day 3-4; (4) Maturity of osteoclasts, i.e.4-6. It was found that there existed three types of filopodia in the osteoclast precursors and only one type of filopodia in the undifferentiated cells. The mature osteoclasts were totally devoid of filopodia. The cell fusion is highly specific, and the fusion first occurs to the filopodia. Filopodia and podosomes not only play important roles in the cell migration and formation of sealing zones, but also in the pre-fusion selectivity of two cells and the movement direction of cell nucleus and cytoplasm during the fusion process. In addition, Cdc42v1, RhoU and RhoF regulate the formation of three different types of filopodia during various stages of differentiation, while Racl, Rac2 and FLNA might be associated with the selectivity of cell during the fusion process.3. The regulatory mechanisms of osteoclast differentiationThe varous changes and the relevant regulatory mechanisms of osteocat differentiation from RAW264.7 were studied using Hoffman microscopy, immunofluorescence staining, DNALadder for agarose gel electrophoresis and qRT-PCR. It was found that except a few RAW264.7 cells that were differentiated into osteoclasts, nearly all undifferentiated RAW264.7 cells experienced apoptosis under the induction of RANKL and M-CSE The RANKL and M-CSF treatments for BRL-3A that had not differentiation ability did not induce BRL-3A apoptosis. When OPG completely inhibited the differentiation of RAW264.7 cells to osteoclasts, apoptosis did not occur to RAW264.7 in spite of the action of RANKL and M-CSF. Racl, RhoA and RhoV are apoptosis-related genes in Rho GTPases. The mRNA expression of RhoV was significantly upregulated, while apoptosis occurred to a large amount of monocytes. However, when the apoptosis of monocytes was inhibited by OPG by inhibiting its differentiation, the RhoV expression was significantly downregulated. On the contrary, the Racl and RhoA expressions did not show variations corresponding to the apoptosis of the monocytes. To sum up, the differentiation of RAW264.7 to osteoclasts caused the apoptosis of RAW264.7. OPG inhibited the RAW264.7 differentiation to osteoclasts, and thereby inhibited the apoptosis of RAW264.7 caused by such differentiation. In the osteoclast differentiation, RhoV mediated the apoptosis of RAW264.7 caused by its differentiation.4. OPG inhibit osteoclast differentiation by affecting the filopodia of RAW264.7Filopodia plays an critical role in the migration and integration of cells. In this study, we investigated the differentiation of osteoclasts and the influence of filopodia and related genes mRNA expression in the precursor osteoclasts induced by OPG. We found that OPG treatment could shorten to filopodia and increase the number of it in the precursor osteoclasts. OPG affected the number and size of osteoclasts in the precursor osteoclasts. The relevant detection conducted precursor cells of osteoclast showed that Cdc42, RhoU, RhoF, Arhgef8/Netl and DOCK5 were down regulated significantly. These data demonstrated that OPG could affect the expression of the Arhgef8/Netl, DOCK5, Cdc42, RhoU and RhoF in RhoGTPases signal pathways, in turn influenced the morphology of filopodia in precursor cells osteoclasts, in the end suppressed osteoclastogenesis.5. The mechanisms of osteoclast bone resorption inhibited by osteoprotegerinThe sealing zone is an osteoclast-specific cytoskeletal structure, the integrity of which is critical for osteoclast-mediated bone resorption. In this study, the sealing zone formation and the effects of OPG on the existing sealing zone and osteoclast-mediated bone resorption activity as well as the regulatory role of genes involved in sealing zone formation were studied by immunofluorescence staining, HMC microscopy, fluorescence quantitative PCR, Western blot and scanning electron microscopy. The sealing zone was formed on Day 5, with belt-like protuberances at the cell edge, scattered distribution of cell nuclei but no filopodia. The sealing zone was intact in the untreated control group. However, defects in the sealing zone were observed in the OPG-treated group (20 ng/mL) and the structure was absent in the groups treated with OPG at 40 ng/mL and 80 ng/mL. The podosomes showed scattered or clustered distribution at the bottom of the osteoclasts. Furthermore, resorption lacunae were not detected in the 20 ng/mL OPG-treated group, indicating loss of osteoclast-mediated bone resorption activity.OPG treatment resulted in significantly decreased expression of Arhgef8/Netl and DOCK5 in Rho GEFs,10 of18 Rho GTPases (RhoA, RhoB, Cdc42v1, Cdc42v2, RhoU/Wrch1, RhoF/Rif, Rac2, RhoG, Rnd1 and RhoBTB1), ROCK1 and ROCK2. In conclusion, podosome distribution was affected by OPG-induced inhibition of the expression of genes in the Rho GTPase signaling pathway. This resulted in damage to or destruction of the sealing zone, thus inhibiting osteoclast-mediated bone resorption activity. |
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