Effects Of Bortezomib On Osteoclastogenesis Of Patients With Multiple Myeloma And The Underlying Machanisms

Many clinical manifestations of multiple myeloma(MM) are associated with loss of bone due to osteolysis,characterized by severe bone pain,pathologic fractures,spinal cord compression and hypercalcemia.Over 85%of MM patients have bone diseases.These devastating events may substantially reduce functional independence and quality of life and pathologic fractures correlate with reduced survival of patients with MM.The phenomenon of increased bone resorption in MM presents the imbalance of bone remodeling in bone marrow microenvironment,which is originally a shift mechanism of mature bone tissues under physiological conditions.The basic process of bone remodeling includes a strong coordination between bone formation managed by osteoblasts and bone degradation managed by osteoclasts,which ensures bone homeostasis and normal growth and development of individuals.Under pathological conditions,the balance is broken due to increased activation of osteoclasts,leading to exacerbation of bone degradation and formation of lytic bone lesions。Excessive activation of osteoclasts in osteolytic bone disease(OBD) of MM may result from stimulation by various cytokines called osteoclast activating factors(OAFs) produced by tumors and stromal cells.The most common OAFs include interleukin-6 (IL-6),IL-1,tumor necrosis factorα(TNF-α),TNF-β,macrophage inflammatory protein 1α(MIP-1α),vascular endothelial growth factor(VEGF) and receptor activator of nuclear factor kappa B(RANK) system.These soluble factors enhance the function of osteoclasts and promote differentiation of osteoclast precursors,and osteoclasts in turn stimulate tumor growth and spread.The vicious circle between enhanced tumor growth and increased bone resorption results in exclusively osteolytic bone lesions,and then pathologic fracture associated with a significant increase in risk of death.Osteoclast activating factors particularly include receptor activator of NF-κB ligand(RANKL),a TNF family member,which was found to be increased in the bone marrow microenvironment in MM.RANKL was shown to be expressed on stromal cells,osteoblasts and myeloma cells.It acts through a trans-membrane receptor RANK which is expressed on osteoclastic lineage cells,while osteoprotegerin(OPG) blocks the direct contact by acting as a soluble decoy receptor for RANKL.RANKL plays a critical role in osteoclast differentiation and regulation.When acting with macrophage-colony stimulating factor(M-CSF),it can induce osteoclast differentiation from bone marrow cells or human PBMCs,even in the absence of osteoblastic stromal cells and other osteotropic factors.RANK lacks intrinsic enzymatic activity in its intracellular domain.Analyses of molecules associated with the cytoplasmic domain of RANK revealed that it transduces signaling by recruiting TNF-receptor associated cytoplasmatic factors(TRAFs). predominantly TRAF-6.As a multifunctional second messenger activated by RANKL. TRAF6 is critical for RANK-induced activation of NF-κB.In vivo experiments showed that TRAF6 gene knocked developed severe osteopetrosis due to impaired bone resorption. It has been recently shown in the context of osteoclastogenesis that TRAF6 forms a signaling complex containing RANK and TAK-1-binding protein(TAB)2,resulting in TGF-β-activated kinase(TAK)1 activation,and then activation of NF-κB,AP-1 and p38 pathways,which are crucial for osteoclast differentiation,survival,and function.Ubiquitin ligase activity mediated by RING finger motif of TRAF6(Lys-63-linked auto-ubiquitination) is important for its ability to activate downstream fcctor.As a member of the proteasome inhibitors' family,bortezomib(VELCADE^TM; formerly PS-341) is the first peptide boronate to enter clinical trials.Many studies have demonstrated its antitumor activity in a variety of tumor types,especially in MM.In the ubiquitin-proteasome pathway,Lys-48-linked poly-ubiquitination marks proteins for degradation by the proteasome,which confers specificity on protein degradation in cells' cycles.Among these proteins IκB is most notable,Phosphorylation, ubiquitination and thereby degradation by proteasome of IκB can release transcription factor NF-κB,which binds its consensus sequences within the promoters of genes encoding cytokines,cell-adhesion molecules and anti-apoptotic proteins.As bortezomib specifically inhibits 26S proteasome,NF-κB has been logically considered to be a key target for bortezomib-induced apoptosis,whose mechanisms include NF-κB inhibition through reduced IκB degradation,leading to reduced NF-κB-dependent synthesis of anti-apoptotic factors such as c-Flip,inhibitor of apoptosis(IAP)1/2,and Bcl-2,and angiogenic factors.The above-mentioned mechanism has been demonstrated in myeloma cells.However,the inhibition of NF-κB by bortezomib could not fully explain anti-tumor-effects mediated by bortezomib.Compared with the specific IKK inhibitor, Hideshima et al found that bortezomib was more potent than the specific IKK inhibitor PS-1145,although both abolished NF-κB activation,suggesting that other signaling pathways or molecules may be targeted by bortezomib.In addition to their anticancer properties,bortezomib and related drugs modulate inflammatory and immune responses by affecting the function and survival of immune cells such as lymphocytes and dendritic cells.In this article we hypothesized that the proteasome inhibitor bortezomib might abolish osteoclast differentiation and functioning, and attempted to find out the relationship between bortezomib and TRAF6,the upper signal factor of NF-κB in osteoclasts.PartⅠ.Generating osteoclasts from human peripheral blood mononuclear cellsTo obtain highly production of osteoclasts from human peripheral blood in enough amount for the study of the mechanism of bone resorption in vitro.The classical osteoclastic induction method from peripheral blood combined with 0.25%trypsin/0.02% EDTA digestion was used to form osteoclast-like cells in the presence of M-CSF and RANKL.A large number' of osteoclasts were induced and produced.The osteoclasts were positive for the TRAP staining and capable of bone resorption in vitro.Conclusion:This method can produce highly production of osteoclasts in enough amount for biochemical and molecular biological research.It is a simple and economical method contrast to other method generating highly purified osteoclasts.PartⅡ.The effects of bortezomib exerts on osteoclastogenesis of MM patients.To observe the effect of bortezomib on the receptor activator of NF-κB ligand (RANKL) mediated osteoclast differentiation and function in vitro.Osteoclast precursors from peripheral blood mononuclear cells of MM patients were cultured in the presence of RANKL and M-CSF.Osteoclast function was quantified with the extent of the ivory slice resorption and TRAP activity in culture supernatants.Sub-apoptotic concentrations of bortezomib used are 0.5nM,1nM,2.5nM and 5nM.Bortezomib can result to reduction of osteoclast differentiation by the detected less formation quantity of osteoclast and the decreased activity level of TRAP in treatment group.Osteoclast resorption capacity was decreased too,reflecting bortezomib can inhibite function of osteoclast.Conclusion:Bortezomib acts on osteoclast differentiation and function at low concentrations and should be considered as potential drug for the treatment of myeloma bone disease.PartⅢ.Machanisms of bortezomib on osteoclastogenesis of MM patientsTo observe the relationship between bortezomib and the upper signal factor of the receptor activator of NF-κB ligand(RANKL) mediated osteoclast TRAF6.Osteoclast precursors from peripheral blood mononuclear cells of MM patients were cultured in the presence of RANKL and M-CSF.Sub-apoptotic concentrations of bortezomib used is 5nM. Using the methods of western-blot and RT-PCR,we observed the changes of bortezomib on protein and mRNA levels of TRAF6.The inhibition of bortezomib on osteoclasts is involved in the decreased production of TRAF6 both in protein and mRNA level. Bortezomib can not inhibit the ubquitination and then degradation of TRAF6.Conclusion:Bortezomib acts on osteoclast differentiation and function at low concentrations through TRAF6 and should be considered as potential drug for the treatment of myeloma bone disease.