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. 2022 Feb 19;28(1):22.
doi: 10.1186/s10020-022-00449-w.

Phosphorylation of BCL2 at the Ser70 site mediates RANKL-induced osteoclast precursor autophagy and osteoclastogenesis

Dianshan Ke #  1   2 , Yunlong Yu #  1   2 , Chenglong Li #  3 , Junyong Han  4 , Jie Xu  5   6
Affiliations

Phosphorylation of BCL2 at the Ser70 site mediates RANKL-induced osteoclast precursor autophagy and osteoclastogenesis

Dianshan Ke et al. Mol Med. .

Abstract

Background: Phosphorylation modification of BCL2 is involved in receptor activator of nuclear factor-κB ligand (RANKL)-induced autophagy of osteoclast precursors (OCPs) and osteoclastogenesis. As an antiapoptotic molecule, the role of BCL2 phosphorylation in osteoclastogenesis is unknown. This study aimed to explore how BCL2 phosphorylation at specific sites regulates osteoclastogenesis.

Methods: We first examined the effects of RANKL on BCL2 phosphorylation at different sites (Ser70 and Ser87) in OCPs. In vivo, transgenic mice overexpressing RANKL (Tg-hRANKL mice) were used to observe the effects of RANKL on phosphorylated BCL2 at different sites in OCPs of trabecular bone. Subsequently, using site-directed mutagenesis, we observed the respective effect of BCL2 mutations at different phosphorylation sites in OCPs on osteoclastogenesis, apoptosis, autophagy and the affinity between BCL2 and Beclin1/BAX under RANKL intervention.

Results: RANKL promoted BCL2 phosphorylation at the Ser70 (S70) site, but not the Ser87 (S87) site, in OCPs. Moreover, Tg-hRANKL mice had stronger BCL2 phosphorylation capacity at S70, not S87, in the OCPs of trabecular bone than wild-type mice in the same nest. Furthermore, BCL2 mutation at S70, not S87, inhibited RANKL-induced osteoclast differentiation and bone resorption activity. In addition, BCL2 mutation at S70 promoted OCP apoptosis, while BCL2 mutation at S87 showed the opposite effect. Remarkably, the BCL2 mutation at S70, not S87, inhibited OCP autophagic activity. Furthermore, BCL2 mutation at S70 enhanced the coimmunoprecipitation of BCL2 and Beclin1, whereas BCL2 mutation at S87 enhanced the coimmunoprecipitation of BCL2 and BAX in OCPs. More importantly, OCP autophagy, osteoclast differentiation and resorption pits inhibited by BCL2 mutation at S70 could be reversed by Beclin1 upregulation with TAT-Beclin1.

Conclusion: RANKL activates OCP autophagy through BCL2 phosphorylation at S70, thereby promoting osteoclastogenesis, which indicates that the inactivation of BCL2 at S70 in OCPs may be a therapeutic strategy for pathological bone loss.

Keywords: Autophagy; BCL2 phosphorylation; Osteoclast; RANKL; Ser70; Ser87.

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Conflict of interest statement

The authors have no competing interests to disclose.

Figures

Fig. 1
Fig. 1
RANKL promoted BCL2 phosphorylation at S70, not S87, in OCPs. A OCPs were treated with the indicated concentration of RANKL for 30 min in α-MEM with 1% FBS (appropriate starvation for enhancing the phosphorylation effect). Phosphorylated BCL2 at S70, i.e., p-BCL2 (S70), and phosphorylated BCL2 at S87, i.e., p-BCL2 (S87), were detected using western blotting assays. B Dynamic changes in p-BCL2 (S70) and p-BCL2 (S87) in OCPs. The protein expression level was normalized to that of the control samples (5 ng/mL). C OCPs were treated with or without 100 ng/mL RANKL for the indicated times in α-MEM with 1% FBS. p-BCL2 (S70) and p-BCL2 (S87) were detected using western blotting assays. D, E Dynamic changes in p-BCL2 (S70) and p-BCL2 (S87) in OCPs. The protein expression level was normalized to that of the control samples (0 min). At different time points, pairwise comparisons were made between the RANKL group and the control group. The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001, **P < 0.01. ns not significant, Cont the control group without RANKL intervention
Fig. 2
Fig. 2
Tg-hRANKL mice had stronger BCL2 phosphorylation capacity at S70, not S87, in OCPs. A Representative 3D micro-CT reconstructed images of the tibiae from Tg-hRANKL mice and control mice in the same nest (WT mice) showing bone mass and bone microstructure (N = 6/group). Scale bar, 2 mm or 1 mm. B Representative H&E-stained tibial sections from each group. Scale bar, 20 μm. C Representative TRAP-stained tibial sections from each group (Red arrows indicate TRAP+ cells). Scale bar, 5 μm. D Tibial sections were stained with red and green fluorochromes for p-BCL2 (S70 or S87) and RANK, respectively, and observed using fluorescence microscopy. The overlapping staining of p-BCL2 and RANK are indicated with red arrows (yellow fluorescence). Scale bar, 2.5 μm. E Representative fluorescent images of p-BCL2 (S70 or S87) (red arrows) in bone marrow RANK+ CSF1R+ cells sorted by FACS. Scale bar, 20 μm. F–K The trabecular bone parameters, including BMD, BV/TV, BS/BV, Tb.Th, Tb.N, and Tb.Sp were analysed using micro-CT. L The trabecular bone parameter, Tb.Ar, was analysed using H&E staining and IPP system. M The number of osteoclasts per millimeter of trabecular bone surface was counted. (N) The percentages of p-BCL2 (S70)-positive cells or p-BCL2 (S87)-positive cells in E (30 cells per field, N = 5). The experiments were replicated at least three times. Data are presented as the mean ± SEM. ***P < 0.001, **P < 0.01. ns not significant, WT control mice in the same nest
Fig. 3
Fig. 3
BCL2 mutation at S70, not S87, inhibited RANKL-induced osteoclastogenesis. A The working model diagram of site-directed mutagenesis: mutation from S70 to S70A (T-G MUT) or mutation from S87 to S87A (AG-GC MUT). B BCL2 expression in OCPs stably transfected with blank vector, wild-type (WT) BCL2, and two mutant BCL2 constructs (S70A or S87A) was detected using western blotting assays. C After OCPs transfected with WT BCL2 and the two mutant BCL2 constructs were treated with or without RANKL for 20 min in α-MEM with 1% FBS, p-BCL2 (S70), and p-BCL2 (S87) were detected using western blotting assays. D The transfected OCPs were treated with M-CSF plus RANKL for 4 days in α-MEM with 5% FBS. Representative images of TRAP-positive multinucleated cells in each group. Scale bar, 200 μm. E The osteoclastic bone resorption activity caused by OCPs inoculated on bone discs and treated with M-CSF plus RANKL for 6 days in α-MEM with 5% FBS was evaluated by scanning electron microscopy. Scale bar, 400 μm. F Quantitative results showing the number of TRAP-positive multinucleated cells in D. G Quantitative results showing the mean resorption pit area in E. The resorption pit area was normalized to that of WT OCPs. H After the transfected OCPs were treated as described in D, the protein expression of CTSK, MMP9, and TRAP was detected using western blotting assays. The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001. ns not significant, Cont the control group without RANKL intervention
Fig. 4
Fig. 4
BCL2 mutation at S70 promoted OCP apoptosis while BCL2 mutation at S87 was contrary. A OCPs transfected with WT BCL2 or S70A BCL2 were treated with RANKL for the indicated times in α-MEM with 2.5% FBS. Cleaved caspase3 and cleaved PARP were detected using western blotting assays. B, C Dynamic changes in cleaved caspase3 and cleaved PARP in OCPs between the WT group and the S70A group. The protein expression level was normalized to that of the control samples (4 h in the WT group). D OCPs transfected with WT BCL2 or S87A BCL2 were treated with RANKL for the indicated times in α-MEM with 2.5% FBS. Cleaved caspase3 and cleaved PARP were detected using western blotting assays. E, F Dynamic changes in cleaved caspase3 and cleaved PARP in OCPs between the WT group and the S87A group. The protein expression level was normalized to that of the control samples (4 h in the WT group). B, C, E, F At different time points, pairwise comparisons were made between the WT group and the mutant group. G The transfected OCPs were treated with RANKL for 48 h in α-MEM with 2.5% FBS. Cell apoptosis was examined by flow cytometry of Annexin/PI staining. H The percentages of apoptotic cells (ANNEXIN-positive cells) are shown in the histograms according to the results in G. The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05
Fig. 5
Fig. 5
BCL2 mutation at S70, not S87, inhibited the autophagic activity of OCPs. A OCPs transfected with WT BCL2 and the two mutant BCL2 were treated with RANKL for 8 h together with or without lysosomal protease inhibitor (E64d plus pepstatin A) in α-MEM with 2.5% FBS. LC3 protein level was detected using western blotting assays. LC3 conversion rate is expressed as the ratio of LC3II to LC3I. B The transfected OCPs were treated with RANKL for 24 h in α-MEM with 2.5% FBS. The autolysosomes (red arrows) in OCPs were observed under TEM. Scale bar, 5 μm or 1 μm. C The histograms showing quantitative results of autolysosomes in B (75 cells from 3 independent experiments). The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001. ns not significant, E E64d, P pepstatin A
Fig. 6
Fig. 6
BCL2 mutation at S70 enhanced the coimmunoprecipitation of BCL2 and Beclin1 in OCPs. A–C The transfected OCPs were treated with RANKL for 2 h in α-MEM with 1% FBS. Cell lysates were extracted for Co-IP with anti-BCL2, anti-Beclin1 or anti-BAX antibody, and subsequently, precipitates were observed using western blotting assays with anti-Beclin1, anti-BAX or anti-BCL2 antibody, respectively. D The transfected OCPs were treated with RANKL for 24 h in α-MEM with 2.5% FBS. The mitochondrial membrane potential was detected by measuring the ratio of red/green fluorescence intensity using JC-10 kit, and quantitatively analysed using flow cytometry. E The percentages of cells in each quadrant are shown in the histograms according to the results in D. The larger proportion of cells in H2 quadrant indicates that mitochondrial membrane potential is higher; The larger proportion of cells in H4 quadrant indicates that mitochondrial membrane potential is lower. The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001. IP the antibody for immunoprecipitation, IB the antibody for immunoblot
Fig. 7
Fig. 7
OCP autophagy and osteoclastogenesis inhibited by BCL2 mutation at S70 was reversed by TAT-Beclin1. A OCPs transfected with WT BCL2 or S70A BCL2 were treated with RANKL along with TAT-Beclin1 (10 μM) or the control peptide (TAT-scrambled) for 8 h in α-MEM with 2.5% FBS. LC3, cleaved PARP, and p62 (soluble and insoluble p62) were detected using western blotting assays. LC3 conversion rate is expressed as the ratio of LC3II to LC3I. B The transfected OCPs were treated with RANKL along with TAT-Beclin1 or TAT-scrambled for 24 h in α-MEM with 2.5% FBS. The autolysosomes (red arrows) in OCPs were observed under TEM. Scale bar, 5 μm or 1 μm. C The histograms showing quantitative results of autolysosomes in B (75 cells from 3 independent experiments). D The transfected OCPs were treated with RANKL along with TAT-Beclin1 or TAT-scrambled for 48 h in α-MEM with 2.5% FBS. Cell apoptosis was examined by flow cytometry of Annexin/PI staining. E The percentages of apoptotic cells (ANNEXIN-positive cells) are shown in the histograms according to the results in D. F The transfected OCPs were treated with M-CSF plus RANKL along with TAT-Beclin1 or TAT-scrambled for 4 days in α-MEM with 5% FBS. Representative images of TRAP-positive multinucleated cells in each group. Scale bar, 200 μm. G, H Quantitative results regarding mature osteoclasts (more than 3 nuclei) or large osteoclasts (more than 5 nuclei) in F. The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001, **P < 0.01
Fig. 8
Fig. 8
The schematic diagram representing the current working model in our study. In brief, BCL2 phosphorylation at Ser87 can dissociate the BCL2-BAX complex, subsequently making proapoptotic molecule BAX lead to apoptotic signal transduction and promote apoptosis, which is not conducive to the differentiation of OCPs into mature osteoclasts. BCL2 phosphorylation at Ser70 can dissociate the BCL2-Beclin1 complex, enabling autophagic molecule Beclin1 to enter autophagic flux and activate autophagy, which represses apoptosis and is conducive to osteoclastic differentiation. RANKL is a stimulator of BCL2 phosphorylation at Ser70, but not Ser87. Therefore, RANKL can induce osteoclastogenesis by phosphorylating BCL2 at Ser70. P phosphorylation, OCP osteoclast precursor, OC osteoclast
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