The Effect And Mechanism Of FKBP51 On Osteoclast Differentiation

Objectives:Osteoporosis is a common bone metabolic disease characterized by decreased bone density,bone microstructural destruction and increased bone fragility.With the aging of the population,the prevalence of osteoporosis continues to rise,becoming a serious health hazard.Abnormalities in the number and/or function of osteoclasts are important pathological mechanisms of osteoporosis.RANKL/RANK/OPG is the most important regulator/receptor of osteoclast differentiation,which participates in osteoclast proliferation,differentiation and maturation through signaling pathways such as NF-?B(nuclear factor-kappa B)and MAPKs(mitogen-activated protein kinases).FKBP51 belongs to a member of the immunophilin family with a complex and extensive cellular biological role.It plays an important regulatory role in stress,tumor,hormone-dependent diseases,reproduction,lipometabolism and immunity.FKBP51 is also involved in the regulation of NF-?B and AKT signaling pathways.Our previous study found that mutant FKBP51V55L could enhance osteoclast differentiation and bone resorption activity,then subsequently participated in the development of Paget bone disease.Moreover,it had been found that the expression of FKBP51 mRNA in osteoclast precursor cells was significantly increased in patients with RA(rheumatoid arthritis).In addition,FKBP5 1 promoted the differentiation and maturation of osteoclasts by up-regulating the expression of FKBP51,but its molecular mechanism is still unclear.The aim of this study was to investigate the effects of FKBP51 on osteoclast differentiation and its molecular mechanisms to provide new molecular targets for the prevention and treatment of osteoporosis.Methods:1.The effect of FKBP51 on osteoclast differentiation:RAW264.7 cells were infected with FKBP51 lentivirus or transfected with FKBP51 interference plasmid and its control to establish FKBP51 overexpression or knockdown cells.The cells were induced with RANKL(50 ng/ml)for 2-3 days,and then assayed by tartrate-resistant acid phosphatase(TRAP)staining.The multi-nucleated cells with positive TRAP staining and more than 2 nucleus were counted to further compare the difference in osteoclastogenesis between the experimental group and its control group.2.Real-time PCR:RT-PCR was used to detect osteoclast-specific molecular CTR,CTSK,OSCAR and osteoclastogenesis-related transcription factors c-Fos,NFATc 1,PU.1,MITF,TRAF6,BLIMP 1 mRNA expression,to further verified the effect of FKBP51 on the differentiation of RAW264.7 cells into osteoclasts at the molecular level.3.Western blot:The protein expression levels of NFATcl,c-Fos and the phos-phorylation levels of AKT,NF-?B,MAPKs(including P38,JNK,ERK)signaling pathway were analyzed by western blot to explore the mecha-nism by which FKBP51 regulated osteoclast differentiation.Results:1.Establishment of FKBP51 overexpression and knockdown RAW264.7 cells:The protein expression level of FKBP51 in FKBP51 overexpression group was significantly higher than its control group(P<0.05);The protein expression level of FKBP51 in FKBP51 knockdown group was significantly lower than its control group(P<0.05),indicating that FKBP51 overexpression and knockdown models was successfully constructed.2.FKBP51 promoted the differentiation of RAW264.7 cells into osteoclasts:Under the induction of RANKL,the number of osteoclasts in FKBP51 overexpression group was significantly higher than its control group(P<0.05),on the contrary,the number of osteoclasts in FKBP51 knockdown group was significantly lower than its control group(P<0.05),indicating that FKBP51 promoted the differentiation of RAW264.7 cells into osteoclasts.3.FKBP51 overexpression enhanced the expression of osteoclast-specific molecules:Compared with the control group,the mRNA expression levels of CTSK,CTR and OSCAR in FKBP51 overexpressing group were increased(P<0.05),which further demonstrated that FKBP51 overexpression promoted the differentiation of RAW264.7 cells into osteoclasts at the molecular level.4.FKBP51 overexpression enhanced the expression of osteoclastogenesis-related transcription factors:During the differentiation of FKBP51 overexpressing cells and controls induced by RANKL,the early protein and mR:NA expression levels of c-Fos and NFATcl were increased(P<0.05),and the mRNA expression levels of other related transcription factors TRAF6,MITF,PU.1 and BLIMP1 were also increased(P<0.05).5.FKBP51 was involved in the regulation of AKT,NF-?B,and MAPKs signaling pathways:Western blot results shown that the phosphorylation levels of AKT Ser473 was downregulated by FKBP51 overexpression,however AKT Thr308 phosphorylation level shown no difference in FKBP51 overexpression and its control group.Meanwhile,NF-?B and MAPKs(including P38,JNK,ERK)phosphorylation levels in FKBP51 overexpression group were elevated(P<0.05).Conclusion:FKBP51 enhanced the expression of key transcription factors c-Fos and NFATc1 by activating NF-?B and MAPKs signaling pathway,and further enhanced the expression of osteoclast-specific molecules CTR,CTSK and OSCAR to promote osteoclast differentiation.