Roles And Molecular Mechanism Of TRPV2 In Attenuating Myeloma Bone Lesions

Multiple myeloma?MM?,also known as plasma cell myeloma,is characterized by excess bone marrow plasma cells.Around the world,MM is the second most frequent hematologic malignancy after non-Hodgkin lymphoma.Approximately 80%of patients with multiple myeloma suffer bone lesions,hypercalcemia,fractures or bone pain during the course of the disease.Myeloma bone disease?MBD?can cause bone destruction and increase the level of Ca²⁺concentration in the bone marrow microenvironment by stimulating osteoclastic differentiation.Previous studies have pointed out that ionized calcium was elevated in the serum and bone marrow microenvironment of MM patients through MM-osteoclast?OCL?interactions by accelerating myeloma bone destruction and reabsorption,which positively correlated with myeloma bone disease and related to hypercalcemic crisis.Transient receptor potential vanilloid 2?TRPV2?,a kind of non-selective ion channels,can be activated by heat,mechanical stress and growth factors.TRPV2 expression is hgher than in cancer cells,compared to normal cells,and with the degree of deterioration of cancer,the expression of TRPV2 also will increase.TRPV2 can regulate cancer cell proliferation,invasion and metastasis by TRPV2 activatior or inhibitors.The thesis is divided into two parts,respectively.?1?Roles of TRPV2 in attenuating myeloma bone disease;?2?TRPV2-induced Ca²⁺-calcineurin-NFAT?signaling regulates differentiation of?osteoclast in multiple myeloma.Here,we reported that the nonselective cation channel transient receptor potential vanilloid 2 plays a functional role in Ca²⁺oscillations and osteoclastogenesis.The blockade of TRPV2 channel may provide a promising treatment strategy in MBD.Part 1 Roles of TRPV2 in attenuating myeloma bone disease1.BackgroundMM cells were exposed to high levels of extracellular calcium([Ca²⁺]_o)in the bone marrow microenvironment surrounding bone destruction.The transportation of calcium ion between inside and outside of cell membrane will influence secretion of inflammatory cytokines.Moreover,the elevated Ca²⁺accelerates myeloma bone destruction and reabsorption through MM-osteoclast?OCL?interactions.Nevertheless,the relationships between MBD and highly efficient stimuli of Ca²⁺in multiple myeloma?MM?progression,and possible regulatory mechanisms are poorly defined.Here,we reported that the nonselective cation channel transient receptor potential vanilloid 2?TRPV2?plays a functional role in Ca²⁺oscillations and osteoclastogenesis.2.ObjectiveThe study was to investigate the expression of TRPV2 channel in multiple myeloma and the relationship with prognosis.We will investigate the change of TRPV2 and osteoclast-related cytokines under high level of extra-cellular calcium concentration;illuminate the correlations between TRPV2 expression levels and osteoclast formation.3.MethodsSerum calcium and clinical indicaters of MM paitients were examined and correlated.At the meantime,to assess whether the dysregulation of serum calcium and bone lesions was related to survival in MM patients,we analyzed public data of MMRF.To assess whether the dysregulation of TRPV channels was detectable in MM patients,we analyzed Pubmed GEO dataset of BM plasma cells from MM,MGUS compared with normal counterparts.Moreover,the distinction between TRPV2expression and survival were analysised by Kaplan-Meier analysis and log-rank tests.Representative image of TRPV2 expression in MM patient was examed by immunehistochemical staining.TRPV2 expression was measured by Western blot in normal and MM patients.Cell viability and apoptosis were detected by trypan blue staining and Flow cytometry with high level of extra-cellular calcium concentration.The Cell counting kit–8 were also determined for detection of cell proliferation.Enzyme-linked immunosorbent assay kits were determined for Receptor Activator of Nuclear Factor-?B Ligand?RANKL?,Interleukin 1 beta?IL-1??and tumor necrosis factor alpha?TNF-??levels.4.Results?1?The levels of TRPV2 in MM plasm cells were higher than normal plasma?NP?cells.Moreover,the distinction between high and low TRPV2 expression were of prognostic significance,as OS were reduced in cases exhibiting higher TRPV2expression in MM patients.?2?Serum calcium is significantly higher in Durie-Salmon?D-S?stage III compared to D-S stages I&II.Similarly,elevated serum calcium was found in International Staging System?ISS?stage III.Moreover,the serum calcium was higher in MM patients with bone lesions compared to those without lesions.Moreover,high serum calcium and bone lesions predict poor prognosis in MM patients.?3?0.4-1.2 mM[Ca²⁺]_o for 12-48 hours?h?showed no differences among the cell growth curves.Thereafter,0.4-1.2 mM were considered as the physiological[Ca²⁺]_o for further epigenetic research.mRNA and protein levels of TRPV2 in LP-1 cells were increased with the increasing concentration of[Ca²⁺]_o,and we also found obvious up-regulation of TNF-?and RANKL after 7d of stimulation with 0.8,1.0 and1.2 mM[Ca²⁺]_o.The expression of TRPV2 was positively correlated with the expression of TNF-?or RANKL.Together,the data demonstrated[Ca²⁺]_o increases the amount of TRPV2 and enhances the secretion of osteoclast-related cytokines in MM cells.5.ConclusionIn conclusion,[Ca²⁺]_o and TRPV2 were high expressed in MM patients,both of them were correlated with poor EFS and OS in MM patients.High concentration of[Ca²⁺]_o could increase the expression of TRPV2 and stimulate the secretion of osteoclast-related cytokines in MM cells.Part2TRPV2-inducedCa²⁺-calcineurin-NFAT?signalingregulates differentiation of?osteoclast in multiple myeloma?1.BackgroundThe occurrence of MBD was related to the secretion of RANKL,TNF alpha,SDF-1,IL-6,IL-3,DKK1,TGF-beta and hepatocyte growth factor?HGF?In pathological conditions and the unbalance of the OCL/OBL differentiation.Previous studies has found that TRP ion channels regulate ion concentration in cellular membranes,which was involved in transcription regulation in cells,leading to occurrence of MBD and metastasis of tumors,and MM cells were exposed to high levels of extracellular calcium([Ca²⁺]_o)in the bone marrow microenvironment surrounding bone destruction,suggesting TRP channels play an important role in the occurrence of MM.Especially,we has found that TRPV2 ion channel was up-regulated and correlated with poor EFS and OS in MM patients.Therefore,it is important to investigate the molecular mechanism of TRPV2 channel on secretion of osteoclast-related cytokines in MM cells and get a new important targets for the treatment of multiple myeloma bone destruction.2.ObjectiveThe study was to investigate the role and molecular mechanism of TRPV2channel on myeloma bone lesions and bone marrow environment.TRPV2 channel may provide a promising treatment strategy in MM and MBD.3.MethodsMM cells cultured in confocal dishes were first loaded with 5?M Fluo 4-AM in Hanks'Balanced Salt Solution?HBSS?at room temperature for 30 min protected from light,then washed twice in HBSS at room temperature.Image analysis was performed using confocal microscopy.SKF96365 and Probenecid were used for the culture of MM cells with extracellular calcium treatment,and then medium and protein were extracted for detection.Up-regulated and knock down the expression of TRPV2 in MM cell,then extraction medium and intracellular proteins used for detection.An enzyme immunoassay kit determined RANKL,IL-1?and TNF-?levels.NFATc3,calcineurin and GAPDH expression were measured by Western blot.Chromatin immunoprecipitation assays were done to reveal that NFATc3 bound to the RANKL promoter.RAW264.7 cells were cultured with MM cells in vitro and staining with TRAP.Numbers of TRAP-positive multinucleated osteoclasts?MNCs???3 nuclei/cell?in the DMSO/SKF96365 groups were counted for osteoclast formation.4.Results?1?The functional regulation of Intracellular Ca²⁺([Ca²⁺]_i)by TRPV2 channel could be blocked by SKF96365;?2?[Ca²⁺]_i could up-regulated calcineurin,which in turn led to NFAT dephosphorylation and nuclear translocation,increase the secretion of osteoclast-related cytokines in MM cells;?3?TRPV2 channel promote the binding of NFATc3 with promoter of RANKL.Inhibition of TRPV2 channel decreased the affinity of NFATc3 and RANKL.?4?The activation and up-regulation of TRPV2 could stimulate the secretion of osteoclast-related cytokines in MM cells.?5?The blockade and knock down of TRPV2 could suppresse myeloma cell-induced osteoclastic differentiation in vitro.5.ConclusionTRPV2 is involved in osteoclastogenesis by activating Ca²⁺-calcineurin-NFATc3signaling pathway,leading to the excessive secretion of inflammatory cytokines and RANKL,which in turn involved in the progression of osteoclastic differentiation.Our results also supported an inhibitory role of SKF96365 in MM-induced osteoclastic differentiation.