scholarly journals The Novel Role of Checkpoint Inhibitor PD-1H/VISTA in Osteoclast Activation and Multiple Myeloma Bone Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1606-1606
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Huihui Ma ◽  
Jun Yang ◽  
Lewis M Brown ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Actin Cytoskeleton ◽  
Trabecular Bone ◽  
Cortical Bone ◽  
Bone Disease ◽  
Checkpoint Inhibitor ◽  
Osteoclast Differentiation ◽  
Bone Lesions ◽  
Osteoclast Activation

Abstract Introduction Multiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed. Methods and Results To identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins. Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts. To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1). Conclusions Taken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Checkpoint Inhibitor PD-1H/VISTA Mediates MMP-13 Induced Osteoclast Activation and Multiple Myeloma Bone Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-16
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Huihui Ma ◽  
Jun Yang ◽  
Gabriel M. Pagnotti ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Trabecular Bone ◽  
Bone Disease ◽  
Checkpoint Inhibitor ◽  
Critical Role ◽  
Private Company ◽  
Osteoclast Activation

Introduction Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues and neurological deficits. MM cells produce osteoclast-activating factors that induce osteoclast activation, thereby leading to excessive bone resorption and lytic bone lesions1. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption via a mechanism independent of its proteolytic activity2. We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed in osteoclasts at high levels3. Binding of MMP-13 to PD-1H/VISTA induces osteoclast fusion and bone resorption activity whereas knockdown or knockout of PD-1H/VISTA largely block MMP-13 mediated effects on osteoclasts3. However, the function of PD-1H inMM bone disease in vitro or in vivo has not been previously defined. Methods and Results To confirm the role of PD-1H in MMP-13 induced bone disease in MM, we first conducted MM-osteoclast trans-well co-culture assay using murine MM cell line, 5TGM1 cells, and bone marrow mononuclear cells from Pd-1h-/- or wild type (WT) mice. 5TGM1 control cells or MMP-13 knockdown 5TGM1 cells were seeded in the upper wells of the transwell plates; while WT or Pd-1h-/- bone marrow mononuclear cells were seeded in the lower wells and cultured for osteoclast differentiation assessed by TRAP staining. Results show that 5TGM1 induced differentiation of WT osteoclasts with significantly increased osteoclast size and nuclei number/osteoclast. Consistent with our previous results2, MMP-13 knockdown blocked the 5TGM1 MM cells-induced activation of WT osteoclasts. In contrast, neither 5TGM1 MM cells nor MMP-13 knockdown cells had significant effects on Pd-1h-/- osteoclasts. Hence, knockout of Pd-1h abrogated MMP-13 mediated MM induction of osteoclasts, indicating that MMP-13/PD-1H interactions are critically involved in MM-induced osteoclast activation. The in vivo role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model2. For this purpose, Pd-1h-/-Rag2-/- mice were generated by crossbreeding C57BL/6 Pd-1h-/- with C57BL/6 Rag2-/- mice. 3x105 firefly luciferase expressing 5TGM1 cells (5TGM1-luc) were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=5). Tumor progression was monitored by weekly bioluminescence imaging (BLI). 3 weeks after tumor inoculation, tibiae were harvested for quantitative micro-CT, followed by histological analysis. Histological staining showed that intratibial injection of 5TGM1-luc MM cells induced extensive lytic lesions and trabecular bone loss in Rag2-/- mice. In contrast, in Pd-1h-/-Rag2-/- mice,the bone structure was maintained with markedly less bone loss. Morphological analyses of trabecular bone across proximal tibiae further indicated that in Rag2-/- mice, 5TGM1 induced significant changes in bone microarchitecture, with decreased bone volume fraction (bone volume/tissue volume), connective density, trabecular bone numbers, and trabecular bone thickness, as well as increased trabecular bone spacing (Table 1). In contrast, in Pd-1h-/-Rag2-/- mice, 5TGM1 failed to induce significant loss of trabecular bone, confirming the critical role of PD-1H in MM induced bone disease in vivo. Conclusions Taken together, our study, for the first time, reveal that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/-mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. References 1. Marino S, Petrusca DN, Roodman GD. Br J Pharmacol. 2019;10.1111/bph.14889. 2. Fu J, Li S, Feng R, et al. J Clin Invest. 2016;126(5):1759-1772. 3. Fu J, Li S, Yang C, et al. Blood. 2019; 134 (Supplement_1): 3072. Disclosures Lentzsch: Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Celularity: Consultancy, Other; Magenta: Current equity holder in private company; Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months.

Brain-Derived Neurotrophic Factor Is a Potential Osteoclast Stimulatory Factor In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1917-1917
Author(s):  
Chun-Yan Sun ◽  
Yu Hu ◽  
Xiao-Mei She ◽  
You Qin ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Disease ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Osteoclast Formation ◽  
Bone Lesions ◽  
Bone Marrow Plasma

Abstract Abstract 1917 Background and Objective: Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In the present study, we investigated whether BDNF was present in marrow from patients with MM and possibly involved in MM cell-induced osteolysis. Methods and Results: Levels of bone marrow plasma BDNF was measured by ELISA in a cohort of individuals with MM and controls. The concentration of BDNF was found to be significantly elevated in patients with MM (879 ± 93) pg/ml when compared with bone marrow plasma derived from normal control subjects (186 ± 52) pg/ml (p < 0.001). Moreover, bone marrow plasma levels of BDNF positively correlated with plasma cell burden and extent of bone disease in MM patients. In osteoclast formation assay, bone marrow plasma from 31 of 37 patients with MM tested significantly stimulated the formation of osteoclast when compared to controls (61.8 ± 7 [mean ± SEM for the 31 patients] versus 25.2 ± 6 TRAP+ multinucleated cells/well [mean ± SEM for the 12 controls]; p < 0.01). The effect was significantly blocked by a neutralizing antibody to BDNF (p < 0.05), suggesting a critical role for BDNF in osteoclast activation. Furthermore, BDNF was found to dose-dependently increased the formation of multinucleated, TRAP+ osteoclast. The direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. Using reverse-transcriptase polymerase chain reaction analysis and western blotting assay, we demonstrated that BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF. These data suggested that TrkB is the functional receptor mediating BDNF's effect on osteoclast formation. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein (MIP)-1α (r = 0.45, p < 0.005) and receptor activator of nuclear factor-κB ligand (RANKL) (r = 0.68, p < 0.0001), two major osteoclast stimulatory factors in MM. Conclusion: Taken together, our results demonstrate the ability of MM cells to secret BDNF correlates with the severity of osteoclastic bone resorption, and provide evidence that BDNF play a causal role in the development of MM bone lesions through TrkB receptor. Disclosures: No relevant conflicts of interest to declare.

Effect of matrix metallopeptidase 13 (MMP13) on multiple myeloma (MM) cells, osteoclast (OCL) activity, and bone resorption.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8099-8099
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Rentian Feng ◽  
Mei Hua Jin ◽  
Farideh Sabeh ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Bone Resorption ◽  
Bone Disease ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Bone Lesions ◽  
Lytic Lesions

8099 Background: MM cells produce OCL-activating factors that induce excessive bone resorption resulting in lytic lesions. The role of MMPs in invasion/progression of solid tumors is well-known, but its function in MM has not been well elucidated. Our group has shown that MMP13 is highly expressed in primary MM cells and in sera of MM patients. Levels of MMP13 significantly correlate with the extent of bone disease. MMP13 is induced by IL-6 via AP-1 activation in MM cells and enhances fusion of OCL precursors resulting in excessive bone resorption. OCL formation using MNCs of mmp-13-/- mice resulted in a fusion defect, significantly decreased OCL size and activity, which could be reversed by exogenous MMP13 (ASH 2009, IMW 2011). Methods: Methods will be presented in the Results section. Results: RT-PCR and western blotting revealed that IL-6 treatment of MM cells induced MMP13 transcription (30-fold) and secretion (>1000-fold). Protein expression of the AP-1 members c-Jun and c-Fos was induced by IL-6, which correlated with MMP13 upregulation. Our data further indicate that the catalytic activity of MMP13 is not required to enhance OCL formation and bone resorption. To prove this, we generated the MMP13 activity-dead mutation MMP13-E223A construct by site-directed mutagenesis PCR-based cloning. The mutated protein was overexpressed in HEK293 cells and purified from the supernatant to confirm whether loss of catalytic activity blocks MMP13 function. To further investigate the in vivo role of MMP13 in MM bone disease, MMP13 expression was knocked down (KD) in murine 5TGM1-MM cells by pKLO. 1 puro lentiviral infection containing sh-RNA targeting mouse MMP13 sequence. MMP13-KD 5TGM1-MM cells or WT-5TGM1-MM cells were intratibially injected into RAG2-/- mice. Development of lytic bone lesions are monitored by micro-QCT and data will be available at the time of presentation. Conclusions: Our data suggest that MMP13, secreted by MM cells, plays a critical role in the development of lytic lesions. Targeting MMP13 represents a promising approach to treat or to prevent bone disease in MM.

scholarly journals Checkpoint Inhibitor PD-1H/VISTA Functions As MMP-13 Receptor on Osteoclasts and Mediates MMP-13 Induced Osteoclast Activation in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3072-3072
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Chen Yang ◽  
Lewis M Brown ◽  
Stephen J Weiss ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Bone Resorption ◽  
Bone Disease ◽  
Research Funding ◽  
Checkpoint Inhibitor ◽  
Clinical Care ◽  
Hek293 Cells ◽  
Bone Lesions ◽  
Binding Interaction

Introduction Multiple myeloma (MM) bone disease is characterized by the development of osteolytic bone lesions due to the over-activation of osteoclast and inhibition of osteoblast cells. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor which is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption by triggering the ERK1/2-dependent up-regulation of the cell fusogen, DC-STAMP. This process operates independently of the MMP-13 proteolytic activity. The fact that MMP-13 regulates OCL signaling via a proteolytic independent mechanism suggests that MMP-13 functions as a paracrine message protein mediating the crosstalk between MM cells and OCL. However, the mechanism is yet to be identified. Methods and Results To screen for MMP-13 cellular binding proteins/receptors, we performed a MMP-13 pull-down assay wherein recombinant MMP-13-His6 was incubated with mouse mononuclear bone marrow cells (BMCs) lysates and Ni-NTA magnetic beads were used to pull-down MMP-13- associated proteins. Following mass spectral analysis, programmed death-1 homolog (PD-1H) was identified as a major MMP-13-binding protein. PD-1H, also known as V-domain Ig suppressor of T cell activation (VISTA), is a critical negative checkpoint regulator expressed on myeloid cells and involved in immune responses. Binding of MMP-13 and PD-1H was confirmed by co-expressing both proteins in HEK293 cells and subsequent co-immunoprecipitation assay. Further, following PD-1H expression in HEK293 cells, an MMP-13-GFP fusion fluorescence protein docked to the cell surface where mutagenesis studies demonstrated that the PD-1H extracellular domain (ECD) mediates the specific binding interaction. Western blot and immunohistochemistry revealed that PD-1H was highly expressed in mononuclear BMCs, pre-OCL and mature OCL. ShRNA-mediated knockdown of PD-1H in mouse mononuclear BMCs blocked the ability of MMP-13 to induce osteoclast fusion and activation. These results were further confirmed by using BMCs from Pd-1h-/- mice and WT littermates for in vitro osteoclast differentiation. While MMP-13 induced WT OCL fusion and activation, these effects were completely blocked in Pd-1h-/-OCLs in tandem with a loss in MMP-13 induced ERK1/2 phosphorylation, NFATc1 and DC-STAMP upregulation. Conclusions Taken together, our study, for the first time, revealed that checkpoint inhibitor PD-1H is highly expressed in pre- and mature osteoclast. More importantly, we identified PD-1H as the critical receptor for MMP-13 in OCL, thereby mediating MMP-13-induced OCL fusion, activation and bone resorption. Hence, MMP-13 as a novel PD-1H ligand might not only induce bone disease, but also play a potential role in the regulation of T-cell activity in MM. As such, targeting MMP-13 and PD-1H interactions may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. Disclosures Lentzsch: Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents & Royalties: 11-1F4mAb as anti-amyloid strategy; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Lumican Inhibits Osteoclastogenesis and Bone Resorption by Suppressing Akt Activity

2021 ◽  
Vol 22 (9) ◽  
pp. 4717
Author(s):  
Jin-Young Lee ◽  
Da-Ae Kim ◽  
Eun-Young Kim ◽  
Eun-Ju Chang ◽  
So-Jeong Park ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Formation ◽  
Map Kinases ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Dual Action ◽  
Dose Dependent ◽  
Resorptive Activity

Lumican, a ubiquitously expressed small leucine-rich proteoglycan, has been utilized in diverse biological functions. Recent experiments demonstrated that lumican stimulates preosteoblast viability and differentiation, leading to bone formation. To further understand the role of lumican in bone metabolism, we investigated its effects on osteoclast biology. Lumican inhibited both osteoclast differentiation and in vitro bone resorption in a dose-dependent manner. Consistent with this, lumican markedly decreased the expression of osteoclastogenesis markers. Moreover, the migration and fusion of preosteoclasts and the resorptive activity per osteoclast were significantly reduced in the presence of lumican, indicating that this protein affects most stages of osteoclastogenesis. Among RANKL-dependent pathways, lumican inhibited Akt but not MAP kinases such as JNK, p38, and ERK. Importantly, co-treatment with an Akt activator almost completely reversed the effect of lumican on osteoclast differentiation. Taken together, our findings revealed that lumican inhibits osteoclastogenesis by suppressing Akt activity. Thus, lumican plays an osteoprotective role by simultaneously increasing bone formation and decreasing bone resorption, suggesting that it represents a dual-action therapeutic target for osteoporosis.

Different responses of trabecular and cortical bone to 1,25(OH)2D3 infusion

1990 ◽  
Vol 259 (5) ◽  
pp. E715-E722
Author(s):  
D. D. Bikle ◽  
B. P. Halloran ◽  
C. McGalliard-Cone ◽  
E. Morey-Holton
Keyword(s):  
Bone Resorption ◽  
Bone Mass ◽  
Trabecular Bone ◽  
Cortical Bone ◽  
Particle Distribution ◽  
Proximal Tibia ◽  
Bone Maturation ◽  
Dihydroxyvitamin D3 ◽  
Per Se ◽  
Bone Particles

Previous studies regarding the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on bone have suggested that 1,25(OH)2D3 increases bone mass and calcium. Many of these studies have focused on trabecular or total bone without examining cortical bone per se. To determine whether the response of trabecular bone to 1,25(OH)2D3 differed from the response of cortical bone, we infused 1,25(OH)2D3 into rats and examined bone mass, 45Ca accumulation, and the density distribution of bone particles (as a measure of bone maturation) in both the proximal tibia and shaft. In the proximal tibia 1,25(OH)2D3 decreased 45Ca accumulation, yet increased bone mass and shifted the particle distribution to more mineralized fractions. In the shaft there was a redistribution of bone to less mineralized fractions that was not accompanied by a change in total bone mass or a decrease in 45Ca accumulation. Thus 1,25(OH)2D3 may retard bone maturation and mineralization throughout the tibia, but this effect in the proximal tibia appears to be overshadowed by a reduction in bone resorption resulting in an accumulation of well-mineralized bone in that region. Bone resorption, however, was not measured directly. The net result is an increase in bone mass and density of trabecular bone not seen in cortical bone.

scholarly journals Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice

2018 ◽  
Vol 238 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Thomas Funck-Brentano ◽  
Karin H Nilsson ◽  
Robert Brommage ◽  
Petra Henning ◽  
Ulf H Lerner ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Bone Formation ◽  
Bone Mass ◽  
Trabecular Bone ◽  
Cortical Bone ◽  
Bone Strength ◽  
Bending Test ◽  
Bone Homeostasis ◽  
Mineral Density

WNT signaling is involved in the tumorigenesis of various cancers and regulates bone homeostasis. Palmitoleoylation of WNTs by Porcupine is required for WNT activity. Porcupine inhibitors are under development for cancer therapy. As the possible side effects of Porcupine inhibitors on bone health are unknown, we determined their effects on bone mass and strength. Twelve-week-old C57BL/6N female mice were treated by the Porcupine inhibitors LGK974 (low dose = 3 mg/kg/day; high dose = 6 mg/kg/day) or Wnt-C59 (10 mg/kg/day) or vehicle for 3 weeks. Bone parameters were assessed by serum biomarkers, dual-energy X-ray absorptiometry, µCT and histomorphometry. Bone strength was measured by the 3-point bending test. The Porcupine inhibitors were well tolerated demonstrated by normal body weight. Both doses of LGK974 and Wnt-C59 reduced total body bone mineral density compared with vehicle treatment (P < 0.001). Cortical thickness of the femur shaft (P < 0.001) and trabecular bone volume fraction in the vertebral body (P < 0.001) were reduced by treatment with LGK974 or Wnt-C59. Porcupine inhibition reduced bone strength in the tibia (P < 0.05). The cortical bone loss was the result of impaired periosteal bone formation and increased endocortical bone resorption and the trabecular bone loss was caused by reduced trabecular bone formation and increased bone resorption. Porcupine inhibitors exert deleterious effects on bone mass and strength caused by a combination of reduced bone formation and increased bone resorption. We suggest that cancer targeted therapies using Porcupine inhibitors may increase the risk of fractures.

scholarly journals Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of Smo in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss

2020 ◽  
Vol 21 (8) ◽  
pp. 2745
Author(s):  
Yukihiro Kohara ◽  
Ryuma Haraguchi ◽  
Riko Kitazawa ◽  
Yuuki Imai ◽  
Sohei Kitazawa
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Signaling Pathway ◽  
Early Stage ◽  
Osteoclast Differentiation ◽  
Osteoclast Formation ◽  
Age Related ◽  
Hh Signaling

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0–48 h (early stage of osteoclast differentiation) or 48–96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.

Knockdown Of Matrix Metalloproteinase 13 (MMP13) In 5TGM1 Multiple Myeloma Cells Inhibits Development Of Lytic Bone Lesions In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 879-879
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Huihui Ma ◽  
G. David Roodman ◽  
Markus Y. Mapara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Trabecular Bone ◽  
Research Funding ◽  
Bone Lesions ◽  
Mineral Density ◽  
Lytic Bone Lesions

Abstract Background Multiple myeloma (MM) cells secrete osteoclastogenic factors that activate osteoclasts (OCL) and contribute to development of pure lytic bone lesions in MM patients. We have recently shown that i) MMP13 is highly expressed by MM cells and ii) exogenous MMP13 increases OCL fusion and bone resorption (Feng et al, 2009). Further, MMP13 mediates these effects by upregulating dendritic cell-specific transmembrane protein (DC-STAMP), which is critical for OCL fusion and activation (Fu et al, 2012). Here, we investigated the role of MMP13 in MM-related bone disease (MMBD) in vivo and the underlying osteoclastogenic mechanisms. Methods and Results The role of MMP13 in MMBD was examined in vivo by the intratibial 5TGM1-GFP mouse MMBD model. Mouse MM cell line 5TGM1-GFP cells were transduced by pLKO.1-puro empty vector (EV) or sh-MMP13 (MMP13-KD) lentivirus followed by puromycin selection for 2 weeks. MMP13 knockdown in 5TGM1-MMP13-KD cells were confirmed by quantitative RT-PCR. 1×105 5TGM1-GFP-EV and 5TGM1-GFP-MMP13-KD cells were bilaterally intratibially injected into Recombination Activating Gene 2 (Rag2) knockout mice (n=9). After 4 weeks of tumor growth, tibiae were separated for micro quantitative computed tomography (micro-QCT) followed by immunohistochemistry (IHC) analysis. Following 5TGM1-GFP-EV injection, micro-QCT analysis of the tibiae and adjacent femurs indicated severe bone erosions, especially within trabecular bone. By contrast MMP13 KD inhibited the development of MM-induced bone lesions. Bone histomorphologic analysis showed that compared to 5TGM1-GFP-EV, MMP13-KD significantly reduced the MM induced trabecular bone loss with increased relative bone volume (0.069 ± 0.018 vs 0.0499 ± 0.016%; P=0.001), connective density (54.94 ± 33.03 vs 27.33 ± 18.97mm3; P=0.002), trabecular bone numbers (3.26 ± 0.29 vs 3.06 ± 0.33mm-1; P=0.032) and bone mineral density (159.1 ± 20.7 vs 134.2 ± 18.6mg/cm3; P=6E-04); as well as decreased triangulation bone surface to volume ratio (66.12 ± 6.67 vs 73.28 ± 10.07; P=0.017) and triangulation structure model index (3.05 ± 0.36 vs 3.42 ± 0.35 mm-1; P=0.002). In accordance with our finding that MMP13 induced OCL fusion, IHC results confirmed the presence of smaller TRAP+OCLs adjacent to the tumor in mice injected with 5TGM1-GFP-MMP13-KD cells compared with 5TGM1-GFP-EV cells. Although MMP13 knockdown showed no effects on 5TGM1-GFP cell growth in vitro, in vivo tumor progression represented by fluorescence imaging and sera immunoglobin 2G level (0.96 ± 0.12 vs 1.10 ± 0.11 mg/ml) was significantly inhibited (P=0.009 and 0.03 respectively), indicating MMP13 depletion in MM cells impaired OCL activation which, in turn, failed to support MM cell growth in bone marrow microenvironment as effectively in EV control group. In vitro studies demonstrated that MMP13 directly induced ERK1/2 phosphorylation in pre-osteoclasts. Consistent with a critical role for ERK1/2 phosphorylation in regulating OCL formation, U0126 (ERK1/2 inhibitor) blocked MMP13-induced ERK1/2 phosphorylation, ERK1/2-dependent DC-STAMP upregulation and MMP13-induced OCL fusion (P<0.01). Conclusion Our results demonstrate that silencing MMP13 expression in MM cells inhibits MM cell-induced OCL fusion and development of lytic bone lesions in vivo, indicating that MMP13 is essential for MM-induced bone diseases. Further, MMP13 upregulates DC-STAMP expression and OCL fusion via the activation of ERK1/2 signaling. Our data suggest that targeting MMP13 may represent a novel therapeutic approach for the treatment of MMBD. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Lilly: Research Funding. Lentzsch:Celgene: Research Funding.

scholarly journals PROGNOSTIC VALUE OF THE BONE TURNOVER MARKERS IN MULTIPLE MYELOMA

2017 ◽  
Vol 39 (1) ◽  
pp. 53-56 ◽  
Author(s):  
D Auzina ◽  
S Lejniece
Keyword(s):  
Multiple Myeloma ◽  
Bone Resorption ◽  
Bone Turnover ◽  
Bone Disease ◽  
Diagnostic Marker ◽  
Control Group ◽  
Bone Lesions ◽  
Bone Resorption Marker ◽  
Baseline Values ◽  
Turnover Markers

Background: Multiple myeloma (MM) is characterized by osteolytic bone disease resulting from increased osteoclast activity and reduced osteoblast function. Aim: The aim of our research was to determine connection between bone turnover markers and presence of bone lesions, their degree of severity, to monitor MM bone disease and to assess effectiveness of anti-myeloma treatment. Materials and Methods: Serum samples and clinical data from 123 patients with newly diagnosed MM were collected at Riga East Clinical University Hospital (Riga, Latvia) from June 2014 to June 2016. Bone lesions detected by radiography, CT scans, MRI, and PET/CT were divided into degrees from 0 to 3 (0 — no bone involvement, 1 — ≤ 3 bone lesions, 2 — ≥ 3 bone lesions, 3 — fracture). Staging was performed applying Durie/Salmon (DS) and International Staging System classifications. Progressive disease was defined as development of one or more new bone lesions. The levels of bone metabolic markers β-isomerized C-terminal telopeptide of collagen type I (β-CTX) and bone-specific alkaline phosphatase (bALP) were monitored regularly in the year. Results: Bone lesions were found in 86 (69%) patients. From these 6 (4%) patients had 1st degree, 11 (9%) had 2nd degree and 69 (56%) had 3rd degree bone lesions. Level of the bone resorption marker β-CTX in the control group was 0.41 ng/ml, which is lower than in MM patients (p < 0.001). Spearman correlation coefficient analysis found a positive and statistically significant correlation (rs = 0.51, p < 0.001) between bone lesions degree and β-CTX levels. Mean β-CTX for patients without bone lesions was 0.72 ng/ml (SD = 0.64), but for patients with 3rd degree bone lesions it was 1.34 ng/ml (SD = 0.65) difference being 38% (p < 0.001). In patients who responded to therapy after 6 months of treatment reduction of β-CTX was found compared to baseline values (M = –0.65). In contrast, in patients who did not respond to therapy, there was a statistically significant (p < 0.001) increase in β-CTX values after six months of treatment compared to baseline values (M = 0.42). Exact cutoff value of β-CTX is 0.79. When analyzing mean bALP, no significant difference between MM patients and control group was found. ANOVA statistical analysis showed no statistically significant differences in bALP levels at different degrees of bone lesions (p = 0.95) in MM patients. Analysis of bALP suitability as MM diagnostic marker using receiver operating characteristics curve showed that bALP is not applicable for clinical diagnosis of MM (AUC 0.5, p > 0.05). However, β-CTX was found to be an excellent diagnostic marker for MM (AUC 0.91; 95% confidence interval, 0.88–0.94; p < 0.001). Conclusions: Patients with MM and bone lesions have increased value of bone resorption marker β-CTX. There is a correlation between bone resorption marker and degree of bone lesions. Changes in β-CTX levels may be used to monitor the effectiveness of myeloma treatment.

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