Effect And Mechanism Study Of Osteoclasts Differentiation And Bone Homeostasis By RNA Binding Protein QKI Deficiency In Myeloid

BackgroundsThe balance of bone metabolism depends on the bone formation and resorption.Any abnormality in the process of bone metabolism will cause a coupling imbalance between bone formation and bone resorption and even lead to a variety of bone disease which will destroy bone microstructure,reduce bone mass,and eventually increase the risk of fracture.As the only widely accepted type of bone resorption cells,osteoclasts play a particularly important role in bone health.Osteoclasts,derived from bone marrow hematopoietic stem cells,are formed by the fusion of multiple monocytes/macrophages.Under the stimulation of M-CSF and RANKL,the corresponding receptors activate the transcription factors by triggering various signal cascades on downstream pathways regulating osteoclasts formation and activation,thus up-regulate osteoclasts functional genes,and promote osteoclasts differentiation and maturation,and finally exert the bone resorption function of osteoclasts.However,our understanding of the specific molecular mechanism of osteoclasts differentiation and maturation is still far from enough at present.RNA binding protein QKI is a member of the STAR family.A large number of studies have shown that QKI plays more important roles in many cell types than initially expected.Recent studies have shown that QKI also has an important effect on the differentiation and polarization of macrophages.Our group previously found that in the mouse model of immune response induced by LPS,QKI was beneficial to the polarization of macrophages to anti-inflammatory type（M2）and reduced the proportion to inflammatory type（M1）.At the same time,we also found that QKI had an inhibitory effect on NF-κB pathway in this model.In view of the significant role of QKI in mononuclear/macrophage lines,we speculate that QKI has a potential function in the process of osteoclasts formation.ObjectiveThe purpose of this study is to explore the role of QKI in osteoclasts formation under bone normal physiological and pathological conditions,and to clarify the molecular mechanism of QKI regulating osteoclasts formation.MethodsSix experiment parts were gradually carried out as below:1.The clinical patients with revised-TKA caused by prosthesis aseptic loosening were chose as the object,and the necrotic bone tissue samples nearby the revision site were collected to detect the expression of QKI.Then the changes of bone metabolism were compared between the Lys M-Cre QKI^（fl/fl）model mice constructed by our group previously and control mice.To explore the effect of QKI lacking in myeloid on bone mass in mice:1)Micro-CT was used to compare the bone mass of control group and KO group.2)the parameters related to bone metabolism（TRAP and OCN）between the two groups were compared using staining and IHC,and 3)the expression levels of ACP5 and OCN in serum between the two groups were compared using ELISA.2.Osteoclasts were induced by primary BMMs from control mice in vitro.Protein and RNA samples were collected regularly to observe the changes of QKI expression during cell induction and to explore the relationship between osteoclasts formation and QKI expression.3.Osteoclasts were induced by primary BMMs from control group and KO group,and the differences of osteoclast differentiation between the two groups were compared.Firstly,the number and area of osteoclasts were compared using TRAP staining.Secondly,the osteoclasts resorption activity was compared using TRAP activity.Finally,the m RNA expression levels of osteoclast biological markers such as Nfatc1,Acp5,Ctsk and Traf6and the protein expression changes of key transcription factors regulating osteoclast differentiation such as NFATc1,C-FOS,TRAF6 were compared using q RT-PCR and Western Blot.4.To explore the specific molecular mechanism of QKI involved in the regulation of osteoclast differentiation,Western Blot was used to compare the expression levels of signaling in classical NF-κB and MAPKs pathways regulating osteoclast differentiation between control group and KO group stimulated by RANKL.5.The causes of osteoblast-osteoclast coupling imbalance in bone homeostasis caused by QKI deletion in myeloid was explored.Firstly,the parameters of IL-1β,TNF-αand RANKL,OPG in the bone marrow of the control group and KO group were compared using IHC,and then the primary BMSCs from the two groups were used to induce osteoblasts using the conditional medium which were consisting supernatants collected from the osteoclasts of the two groups,and the formation of osteoblasts was compared.6.The osteoporosis disease models were established based on the control group and KO group to verify the effect of QKI on bone metabolism under pathological condition.1)Micro-CT was used to compare the bone mass of each group.2)The bone metabolism related parameters（TRAP and OCN）were compared using staining and IHC.3)The expression levels of ACP5 and OCN in serum of each group were compared using ELISA.Results1.The expression level of QKI in revised-TKA group was significantly lower.Then Micro-CT showed that compared with the control group,the bone microstructure of KO group was damaged and the bone mass was significantly decreased（P<0.001）.TRAP staining and OCN immunohistochemistry results showed that the number of TRAP positive cells per unit area of bone tissue in KO group was significantly increased（P<0.01）,while OCN was significantly decreased（P<0.05）.ELISA results showed that the concentration of ACP5 in serum of KO group was significantly higher（P<0.05）,while OCN was significantly decreased（P<0.05）.2.The results of TRAP staining showed that the number and area of osteoclasts increased gradually under the co-stimulation of M-CSF and RANKL.On the 5th day after induction,a large number of typical osteoclasts formed.The results of q RT-PCR and Western Blot showed that the expression levels of biological markers and important transcription factors related to osteoclasts differentiation were significantly increased,meanwhile QKI expression decreased gradually during osteoclasts formation.3.After stimulation with RANKL,the number and the area of osteoclasts in KO group were significantly higher（P<0.0l）.The activity of TRAP in the supernatant of KO group was significantly higher after induction with RANKL（P<0.0l）.The results of q RT-PCR showed that the expression level of osteoclast-related Marker-Acp5,Ctsk,Nfatc1,C-fos and Traf6 in KO group was significantly higher（P<0.0l）.Western Blot results showed that the protein expression of NFATc1,C-FOS,CN in KO group was higher.Besides,the peak expression levels of C-FOS and CN in KO group appeared much earlier.4.After stimulated by RANKL,the NF-κB and MPAKs pathways were activated in different degrees in both two groups.The activities of important isoforms and enzymes of the pathways in KO group increased significantly.The maximum differences were found at 15～30 minutes after stimulation.5.The immunohistochemical results of bone tissue showed that the number of IL-1βand TNF-αpositive cells in KO group was significantly higher（P<0.05）.The number of RANKL positive cells in KO group was significantly higher（P<0.05）,while OPG was significantly lower（P<0.05）.Under the condition of complete medium induction,the positive area of alizarin red staining in KO group was less than that in the control group,and when induced in the conditional medium,the positive area in the two groups was much less than before,especially in KO group.6.Compared with the other two groups,the bone mass of KO mice after OVX was significantly decreased and there was more serious osteoporosis in KO-OVX group（P<0.001）.The results of bone tissue staining and immunohistochemistry showed that the number of TRAP positive cells per unit area of bone tissue in KO group was significantly higher（P<0.001）,while OCN was significantly decreased（P<0.01）.The results of ELISA showed that the ACP5 concentration in serum of KO group was significantly higher（P<0.001）,while the OCN concentration was significantly decreased（P<0.01）.ConclusionsThis study has revealed that QKI deficiency in myeloid can promote osteoclasts differentiation and enhance the bone absorption function by enlarge the cascade of NF-κB and MAPKs pathways related to osteoclasts differentiation,and increasing the proportion of RANKL/OPG.On the other hand,due to the QKI deficiency in myeloid,bone marrow inflammatory microenvironment and osteoclast-osteoblast interaction can inhibit osteoblasts formation and thus disrupt bone balance.Under the influence of the two factors above,it can eventually lead to the decrease of bone mass and increase the tendency of osteoporosis,especially under pathological conditions.We hold the opinion that lack of QKI in myeloid can be regarded as a high-risk factor for bone loss diseases and maintaining QKI expression lever in myeloid has a potential anti-bone resorption effect.