Research On The Role And Mechanism Of PFKFB3 In Osteoclast Differentiation

Objective:Osteoclast is the only cell type responsible for bone resorption in human body.An excessive number or overactive function of osteoclasts is considered to be the major pathological mechanism of osteoporosis.The differentiation of osteoclasts is dependent on the collaborative effects of two essential cytokines,M-CSF and RANKL.Stimulation by RANKL during osteoclastogenesis leads to the activation of NF-?B and MAPK signaling cascades and induces a metabolic shift to elevated glycolytic metabolism.PFKFB3 is an important enzyme with the ability to regulate glycolysis.Suppression of PFKFB3 can reduce the cellular glycolytic activity and inhibit the activation of NF-?B and MAPK pathways.We thus speculated that PFKFB3 might participate in the regulation of osteoclastogenesis.The aim of this study is to investigate the role and the underlying mechanism of PFKFB3 during osteoclast differentiation and provide novel targets and potential therapeutic strategies for osteoporosis and other osteoclast-related disorders.Methods:1.During RANKL-induced osteoclast differentiation from BMMs,the protein levels of PFKFB3 were detected by western blotting,and the glycolytic activity in BMMs was measured using the glucose assay kit and the lactate assay kit.2.The adenovirus carrying PFKFB3 sh RNA or PFKFB3 inhibitor PFK15 was used respectively to reduce the expression of PFKFB3 in BMMs,followed by osteoclast differentiation using RANKL stimulation.TRAP staining was performed to evaluate the differentiation of osteoclasts,F-Actin staining and pit formation assay were conducted to assess the function of osteoclasts.Meanwhile,the expression of osteoclast marker genes and the functional status of NF-?B and MAPK signaling were detected by q PCR and western blotting.3.A post-menopausal osteoporosis murine model was established using ovariectomy surgery.The development of osteoporosis in this model was attributed to the lack of estrogen and the excessive activation of osteoclasts.After 6 weeks of intervention by PFK15,the femurs of the mice were collected for micro-CT scanning and histomorphometric analysis.4.Lactate was used to rescue the inhibitory effects of PFK15 on osteoclastogensis.Osteoclast differentiation was evaluated by TRAP staining.The expression of osteoclast marker genes and the functional status of NF-?B and MAPK signaling were detected by q PCR and western blotting.Results:1.During RANKL-induced osteoclastogenesis,both the protein expression of PFKFB3 and the glycolytic activity represented by glucose consumption and lactate production increased,indicating that PFKFB3 might participate in the regulation of osteoclast differentiation.2.Either the adenovirus carrying PFKFB3 sh RNA or PFKFB3 inhibitor PFK15 significantly inhibited osteoclast differentiation,impaired bone resorption function of osteoclasts and reduced the expression of osteoclast marker genes,suggesting that PFKFB3 is a prerequisite for osteoclast differentiation and function.3.PFKFB3 inhibitor PFK15 attenuated ovariectomy-induced bone loss and reduced osteoclast formation in the ovariectomized murine model,indicating that PFKFB3 can also generate its effects on osteoclastogenesis in vivo.4.Inhibition of PFKFB3 repressed the activation of NF-?B and MAPK signaling,suggesting that the inhibitory effects on osteoclastogenesis caused by PFKFB3 inhibition might rely on the suppression of NF-?B and MAPK pathways.5.Inhibition of PFKFB3 reduced the glycolytic activity represented by glucose consumption and lactate production.Administration of lactate partially reversed the repression of osteoclastogenesis caused by PFKFB3 inhibition and abrogated the inhibitory effects of PFK15 on the activation of MAPK and NF-?B pathways.These results revealed that glycolysis participated in PFKFB3-mediated regulation of osteoclastogenesis.Conclusion:In conclusion,our results demonstrated that inhibition of PFKFB3 suppressed osteoclast differentiation and impaired osteoclastic bone resorption in vitro,and prevented ovariectomy-induced bone loss in vivo.All in all,the results of this study clarified the essential role of PFKFB3 during RANKL-induced osteoclastogenesis and indicated that blockage of glycolysis by targeting PFKFB3 represented a potential therapeutic strategy for osteoporosis and other osteoclast-related disorders.