| Osteoprotegerin (OPG), as a member of the tumor necrosis factor (TNF) receptor superfamily, has been found early in 1990s. OPG is known to inhibit differentiation and activation of osteoclasts (OCs) by functioning as a decoy receptor blocking interactions between Receptor Activator for Nuclear Factor-KB (RANK) and RANKL. Our team found that OPG could induce the apoptosis of mature OCs cultivated in vitro. It might be the main route that OPG suppressed the activity of OCs. However, the exact role of OPG in the survival/apoptosis of OCs remains unclear. Malnutrition in bone in large animal and mass breeding animals have become more and more serious with the further development of animal husbandry industry in China. It becoming more and more important to using the recombinant OPG and OPG gene therapy technology to prevent bone disease in animal. But the inhibition effect of OPG on the survival of OCs have not been well elucidated. All these make it difficult to develop and use OPG related drug to cure the nutrition metabolic bone disease of livestock and poultry. In this study, isolated murine bone marrow borrow were induced to OCs and osteoclast precursor cells (OPCs), different concentrations of OPG were added when the OCs were mature. TACS-XL Blue assay (derivative method of TUNEL apoptosis detection), QRT-PCR, Western blot, Co-IP etc. were all performed to clarify the effect of OPG on the morphological changes, functions, related genes expression, and key signaling proteins of apoptosis in OCs and OPCs. The aim of this study was to determine the precise mechanism of apoptosis induced by OPG in OCs and OPCs. This would provide theoretical basis and new treatment strategy for the development and application of related drugs. All test carried out were showed below.1. OPG induces OCs and OPCs apoptosisIn order to figure out the influence of OPG on the apoptosis of murine OCs and OPCs cultured in vitro, bone marrow cells were harvested from 8-12-week-old male Balb/cJ mice and incubated with M-CSF+RANKL for osteoclastogenesis. Meanwhile, bone marrow cells incubated with only M-CSF were used to induce the OPCs model. After 5 days cultivation, RANKL was removed, different concentration of OPG (0,20,40 and 80 ng/ml) were added. Annexin V-FITC immunofluorescent assay, flow cytometry using Annexin V-FITC and propidium iodide (PI) double staining kit, and TACS-XL Blue assay were performed to determine the early apoptosis and late apoptosis rate of OPCs, apoptotic morphological changes of OCs, respectively. The results showed that, compared to control group, OPG treatment would significantly increase the apoptotic rate of OPCs (P<0.05). The early apoptotic rate and late apoptotic rate reached the peak in 40 ng/mL OPG treatment group. OPG treatment significantly increased the apoptotic rate of OCs in a dose-dependent manner, compared to control group (P<0.05).2. The effect of OPG on apoptotic genes in OCsThe aim of this study was to detect the alteration of apoptotic genes induced by OPG in OCs. Murine bone marrow were incubated with M-CSF+RANKL for osteoclastogenesis. After 5 days cultivation,0,20,40 and 80 ng/ml of OPG were added to different groups of cells, after removing RANKL. OCs were collected, RNA and total protein were isolated, and then levels of gene transcription and protein expression of apoptotic genes were monitored via QRT-PCR and Western blot. The results showed that OPG treatment would significantly promote the transcriptional level of Bax gene (P<0.05), highly significant suppress the transcriptional level of Bcl-2 gene (P<0.01), in dose-dependent manner. Simultaneously, the results of Western blot showed that, compared to control group, the expression of Bax ascended, the expression of Bcl-2 descended, the ratio of Bax/Bcl-2 increased highly significant (P<0.01), according to the increasing of concentration of OPG. These data indicated that OPG induced OCs apoptosis by accelerating the expression of pro-apoptotic genes and inhibiting the anti-apoptotic genes.3. OPG induced OCs and OPCs apoptosis via Fas/FasL pathwayTo determine the molecular mechanism of Fas/FasL signalling pathway involved in apoptosis induced by OPG in OCs and OPCs, murine bone marrow were incubated with M-CSF+RANKL for OC and OPC. After cultivation, OPG of relative concentration and/or the antagonist of Fas/FasL pathway were added to different groups of cells. QRT-PCR and Western blot were used to detect the effect of OPG on the levels of transcription and expression of key signaling proteins involved in Fas/FasL pathway in OCs and OPCs. In addition, the change of soluble FasL (sFasL) concentration in the cell supernatant and the binding of sFasL were detected by ELISA and Co-IP, respectively. The results demonstrated that, compared to control group, the transcription and expression levels of Fas and activated caspase-8 was increased by both 20 ng/mL and 40 ng/mL of OPG, but these were markedly decreased at 80 ng/mL (P<0.05). In the meantime, transcriptional and expression levels of FasL increased with increasing doses of OPG, sFasL in the supernatant significantly decreased (P<0.05). The co-treatment of antagonist of Fas/FasL pathway with OPG would highly significant attenuate the activation of caspase-8 and caspase-3 (P<0.01). Co-IP assay found that sFasL would bind OPG. The binding would block the inhibition of the apoptosis of OCs and OPCs.4. OPG induced OCs and OPCs apoptosis through mitochondrial apoptotic pathwayTo investigate the role of mitochondrial pathway in apoptosis induced by OPG in OCs and OPCs, OCs and OPCs induced by method showed above were treated with OPG with corresponding concentrations. Cells were collected at the end of cultivation, Western blot was used to detect the distribution of cytochrome C (cyt. c), related proteins of mitochondrial apoptotic pathway, activation of caspase-9 and caspase-3. Immunofluorescence staining technique was used to detect nuclear translocation of Apoptosis Inducing Factor (AIF) and Endoribonuclease G (Endo G). The results showed that OPG treatment could induce the release of cyt. c from mitochondrion to cytoplasm, the activation of caspase-8 and caspase-3, and the nuclear translocation of AIF and Endo-G. All these results clarified that OPG would induced OCs and OPCs through mitochondrial apoptotic pathway. |
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