The Role Of JNK1-autophagy Signaling In The Regulation Of RANKL-promoted Osteoclastogenesis

Osteoporosis is a systemic bone metabolic disease characterized by decreasing bone mass,destroying bone tissue microstructures,increasing bone brittleness and aggravating the fracture’risk.Bone remodeling is a highly dynamic process under the control of bone resorption mediated by osteoclasts(OCs)and formation mediated by osteoblasts(OBs).Osteoclast is the major bone resorbed cell that differentiate from monocyte/ macrophage-lineage hematopoietic cells.The number and activity of osteoclasts directly determine the rate of bone turnover,density and structure.RANKL plays a critical role in osteoclastogenesis.Moreover,RANKL can promote osteoclastogenesis via JNK1 signal.The activation of JNK1 can induce the formation and activation of AP-1,a transcriptional factor through phosphorylation of c-Jun,subsequently leading to the OCs formation.Nevertheless,Activated JNK1 can regulate the osteoclastogenesis via c-Jun phosphorylation-independent manner.It has been reported that JNK1 can activate autophagy under stress.Involvement of autophagy in the proliferation and differentiation of OCs as well as the activity of bone absorptionhave been proved;and autophagy was greatly activated in response to RANKL.However,the role of JNK1-mediated autophagy on osteoclastogenesis has not been verified.Transient nutrient deficiency could trigger JNK1-induced low level phosphorylation of Bcl-2,followed by dissociation of Beclin1,altogether promoting autophagy and survival.Besides,Beclin1 plays a significant role during osteoclastogenesis.Regarding the pathway connections between RANKL and JNK1,RANKL was likely to promote Bcl-2 phosphorylation via JNK1 activation,subsequently facilitating Beclin1 into autophagy flux.Therefore,we presumed that JNK1-Bcl-2-Beclin1-autophagy signal pathway might be other mechanism independent from c-Jun phosphorylation during RANKLinduced osteoclastogenesis.In conclusion,the aim of the current study was to elucidate the significance of RANKL-JNK1-Bcl-2-Beclin1-autophagy pathway in osteoclastogenesis,so as to provide more pharmacological targets for clinical treatment of osteoporosis,while enriching the mechanisms of RANKL-induced autophagy.METHODS:Using murine macrophage cell line RAW264.7 and mouse bone marrow-derived macrophages(BMMs)as osteoclast precursors,combined with Western blotting analyses,lentiviral transduction,siRNA transfection,GFP fluorescence detection,electron microscopy,TRAP staining,qRT-PCR,annexin V-FITC/PI staining,Tunel staining,co-immunoprecipitation and co-immunocallocating,We explored the role and the possible mechanism of RANKL-JNK1 signal in the regulation of autophagic activity during the osteoclastogenesis in vitro.In addition,through the research of IL-17 A intervention,the JNK1-mediated autophagy was introduced into the study of inflammatory bone metabolism in order to enrich the mechanism above.RESULTS:Part I.JNK1 regulates RANKL-mediated osteoclastogenesis through autophagy-TRAF3 signaling1.The autophagy protein Beclin1 could rescue JNK1 inhibition-induced osteoclast precursors(OCPs)autophagy inactivation in RANKL-stimulated osteoclastogenesis1)Overexpression of Beclin1 in OCPs increased the LC3 conversion in the presence of JNK inhibitor.2)RANKL enhances the phosphorylation of JNK1,while JNK1 silencing attenuates LC3 conversion in OCPs.3)The number of LC3 punctation by JNK inhibitor were restored by Beclin1 overexpression.4)JNK inhibitor inhibited the formation of autolysosome,which was increased by Beclin1 overexpression.2.Overexpression of Beclin1 could partially restored the reduced osteoclastogenesis by JNK inhibitior3.Knockdown of TRAF3 reversed the reduced osteoclastogenesis mediated by JNK inhibitior1)Beclin1 overexpression enhanced TRAF3 degradation of OCPs in the presence of JNK inhibitor.2)Knockdown of TRAF3 by siRNA reversed JNK1 inhibiton-induced reduction of osteoclastogenesis.4.TRAF3 degradation was not affected by RANKL in the presence of Chloroquine1)There was no difference in TRAF3 experession between RANKL plus chloroquine and single administration of chloroquine.2)A combination of RANKL and chloroquine resulted in an obvious elevated expression of TRAF6 and p-JNK1.5.JNK inhibitors alleviated TRAF3 degradation and enhanced Chloroquine mediated TRAF3 stabilityPart II.JNK1-mediated autophagy prevents apoptosis during osetoclastogenesis via TRAF3 degradation.1.Both Beclin1 overexpression and rapamycin inhibit the apoptosis of OCPs,while chloroquine is contrary.1)The cleavage of caspase-3 and PARP were significantly decreased after Beclin1 overexpression in OCPs.2)The cleavage of caspase-3 and PARP and apoptotic cells were all decreased after adding rapamycin,which was totally converse after administration of chloroquine.2.The inhibition of autophagy in OCPs is accompanied by up-regulation in apoptosis after inhibition of JNK11)JNK1 silencing could promote apoptosis,while preventing RANKL-induced autophagy of OCPs.2)The upregulation of apoptosis induced by SP600125 were significantly decreased by Beclin1 overexpression.3.TRAF3 silencing inhibits apoptosis of OCPsThe expression of cleaved caspase-3 and PARP as well as apoptotic cells were decreased after knockdown of TRAF3 by siRNA in OCPs.4.TRAF3 knockdown inhibits chloroquine-up-regulated OCPs apoptosisThe expression of cleaved caspase-3 and PARP as well as apoptotic cells were significantly inhibited by down-regulation of TRAF3 in OCPs.5.TRAF3 knockdown prevents the enhanced OCPs apoptosis by JNK1 inhibitionThe cleaved caspase-3 and PARP as well as apoptotic cells were decreased after TRAF3 knockdown in the presence of JNK inhibitor.Part III.JNK1-regulated Bcl-2 phosphorylation mediates OCPs ’ autophagy1.There exists association between p-JNK1,p-Bcl-2 and LC3 II in the presence of RANKLAfter induction of RANKL,both the time-dependent or concentration-dependent of p-JNK1,p-Bcl-2 and LC3 II shared a similar trend,and a positive correlation.2.JNK1 inhibition reverses RANKL-promoted Bcl-2 phosphorylationRANKL could raise the level of p-Bcl-2,which reduced once again after upregulation of JNK1.3.JNK1 inhibition reverses the inhibited combination between Beclin1 and Bcl-2 by RANKLRANKL could lead to the attenuated combination of Beclin1 and Bcl-2,which was enhanced again both in the presence of JNK inhibitor or JNK1 silencing.Part IV.IL-17 A regulates the autophagic activity of osteoclast precursors through RANKL-JNK1 signaling1.A low concentration of IL-17 A could enhance OCPs autophagy,whereas a high level of IL-17 A is contrary2.Autophagy inhibitor can attenuate IL-17A-promoted osteoclastogenesis3.JNK signal mediates IL-17A-promoted OCPs autophagyCONCLUSIONS:1.JNK1 signals could mediate RANKL-induced autophagy activation of OCPs and osteoclastogenesis via Beclin1.2.JNK1-mediated autophagy might promote osteoclastogenesis via TRAF3 degradation.3.RANKL-mediated TRAF3 degradation only relies on its downstream pathways to activate autophagy.4.The autophagy activation of OCPs is accompanied by resistance of apoptosis during osteoclastogenesis.5.TRAF3 could mediate the apoptosis,and bridge autophagy and apoptosis in OCPs.6.JNK1-mediated TRAF3 degradation could prevent apoptosis of OCPs.7.RANKL has effect on autophagic activity of OCPs through Bcl-2-phosphorylation.8.A low concentration of IL-17 A is likely to promote autophagic activity via activating RANKL-JNK pathway during osteoclastogenesis.