Abstract
BackgroundPhosphorylation 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.MethodsWe 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.ResultsRANKL 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.ConclusionRANKL 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.
Highlights
Pathological bone loss is a group of diseases related to the skeletal system based on the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, which is characterized by decrease bone mass and damaged bone microstructure (Yu et al 2021; Fujii et al 2021)
Tg‐hRANKL mice had stronger B-cell lymphoma 2 (BCL2) phosphorylation capacity at S70, not S87, in osteoclast precursors (OCPs) We documented the differential effects of Receptor activator of nuclear factor-κB ligand (RANKL) on BCL2 phosphorylation at different sites in OCPs in vitro
We used Tg-hRANKL transgenic mice to observe the in vivo effect of RANKL on BCL2 phosphorylation at different sites in OCPs
Summary
Pathological bone loss is a group of diseases related to the skeletal system based on the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, which is characterized by decrease bone mass and damaged bone microstructure (Yu et al 2021; Fujii et al 2021). A better understanding of the mechanism of RANKL during osteoclastogenesis and finding more efficient and specific therapeutic targets are the keys to the prevention and treatment of pathological bone loss. In addition to the identified c-Jun/AP-1 pathway, there is a BCL2-Beclin1-autophagy activation pathway in JNK1mediated osteoclastogenesis (Ke et al 2019a). JNK1 causes the dissociation of the B-cell lymphoma 2 (BCL2)Beclin complex by the phosphorylation of BCL2, further inducing autophagy activation (Wei et al 2008a), which is found in RANKL-induced osteoclastogenesis (Ke et al 2019a). As a positive factor of Beclin1-dependent autophagy, the role of BCL2 phosphorylation in osteoclastogenesis remains unclear. Phosphorylation modification of BCL2 is involved in receptor activator of nuclear factor-κB ligand (RANKL)-induced autophagy of osteoclast precursors (OCPs) and osteoclastogenesis. This study aimed to explore how BCL2 phosphorylation at specific sites regulates osteoclastogenesis
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