Metformin attenuates osteoclast-mediated abnormal subchondral bone remodeling and alleviates osteoarthritis via AMPK/NF-κB/ERK signaling pathway

This study explored the mechanism by which metformin (Met) inhibits osteoclast activation and determined its effects on osteoarthritis (OA) mice. Bone marrow-derived macrophages were isolated. Osteoclastogenesis was detected using tartrate-resistant acid phosphatase (TRAP) staining. Cell proliferation was evaluated using CCK-8, F-actin rings were detected by immunofluorescence staining, and bone resorption was detected using bone slices. Nuclear factor kappa-B (NF-κB) and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) were detected using luciferase assays, and the adenosine monophosphate-activated protein kinase (AMPK), NF-κB, and mitogen-activated protein kinase (MAPK) signaling pathways were detected using western blotting. Finally, expression of genes involved in osteoclastogenesis was measured using quantitative polymerase chain reaction. A knee OA mouse model was established by destabilization of the medial meniscus (DMM). Male C57BL/6J mice were assigned to sham-operated, DMM+vehicle, and DMM+Met groups. Met (100 mg/kg/d) or vehicle was administered from the first day postoperative until sacrifice. At 4- and 8-week post OA induction, micro-computed tomography was performed to analyze microstructural changes in the subchondral bone, hematoxylin and eosin staining and Safranin-O/Fast Green staining were performed to evaluate the degenerated cartilage, TRAP-stained osteoclasts were enumerated, and receptor activator of nuclear factor κB ligand (RANKL), AMPK, and NF-κB were detected using immunohistochemistry. BMM proliferation was not affected by Met treatment below 2 mM. Met inhibited osteoclast formation and bone resorption in a dose-dependent manner in vitro. Met suppressed RANKL-induced activation of p-AMPK, NF-κB, phosphorylated extracellular regulated protein kinases (p-ERK) and up-regulation of genes involved in osteoclastogenesis. Met reversed decreases in BV/TV, Tb.Th, Tb.N, and CD, and an increase in Tb.Sp at 4 weeks postoperatively. The number of osteoclasts and OARSI score were decreased by Met without effect on body weight or blood glucose levels. Met inhibited RANKL, p-AMPK, and NF-κB expression in early OA. The mechanism by which Met inhibits osteoclast activation may be associated with AMPK/NF-κB/ERK signaling pathway, indicating a novel strategy for OA treatment.

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

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.

Bone Regeneration Using MMP-Cleavable Peptides-Based Hydrogels

Accumulating evidence has suggested the significant potential of chemically modified hydrogels in bone regeneration. Despite the progress of bioactive hydrogels with different materials, structures and loading cargoes, the desires from clinical applications have not been fully validated. Multiple biological behaviors are orchestrated precisely during the bone regeneration process, including bone marrow mesenchymal stem cells (BMSCs) recruitment, osteogenic differentiation, matrix calcification and well-organized remodeling. Since matrix metalloproteinases play critical roles in such bone metabolism processes as BMSC commitment, osteoblast survival, osteoclast activation matrix calcification and microstructure remodeling, matrix metalloproteinase (MMP) cleavable peptides-based hydrogels could respond to various MMP levels and, thus, accelerate bone regeneration. In this review, we focused on the MMP-cleavable peptides, polymers, functional modification and crosslinked reactions. Applications, perspectives and limitations of MMP-cleavable peptides-based hydrogels for bone regeneration were then discussed.

Mixed lineage kinase domain-like pseudokinase mediated necroptosis aggravates periodontitis progression

Necroptosis is a form of cell death that is reportedly involved in the pathogenesis of periodontitis. However, the role of Mlkl-involved necroptosis remains unclear. Herein, we aim to explore the role of MLKL-mediated necroptosis in periodontitis in vitro and in vivo. Expression of RIPK3, MLKL, and phosphorylated MLKL is observed in gingival tissues obtained from healthy subjects or patients with periodontitis. Viability of Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells was detected. In wild type or Mlkl deficiency mice with ligature-induced periodontitis, alveolar bone loss and osteoclast activation were assessed. mRNA levels of inflammatory cytokines in bone marrow-derived macrophages were tested by qRT-PCR. Increased expression of RIPK3, MLKL, and phosphorylated MLKL is observed in gingival tissues obtained from patients with periodontitis. Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells developed necroptosis after caspase inhibition and negatively regulated the NF-κB signaling pathway. In mice with ligature-induced periodontitis, Mlkl deficiency reduced alveolar bone loss and weakened osteoclast activation. Furthermore, genetic ablation of Mlkl in LPS-Pg-treated bone marrow-derived macrophages increased the mRNA levels of tumor necrosis factor-α, interleukin (Il)-1β, Il-6, cyclooxygenase 2, matrix metalloproteinase 9, and receptor activator of nuclear factor kappa-B ligand. Our data indicated that MLKL-mediated necroptosis aggravates the development of periodontitis in a Mlkl-deficient mouse. And this will provide a new sight for the understanding of etiology and therapies of periodontitis.

Antibodies to Citrullinated Proteins (ACPA) Associate with Markers of Osteoclast Activation and Bone Destruction in the Bone Marrow of Patients with Rheumatoid Arthritis

Normalizing bone metabolism is a challenge in rheumatoid arthritis (RA). Studies in mice suggest that anti-citrullinated protein antibodies (ACPAs) can trigger osteoclast activation and bone resorption in the bone marrow. However, data on the presence and role of ACPAs in human bone marrow are scarce. We investigated whether ACPAs can contribute to osteoclast activation and bone erosion in RA bone marrow. Anti-cyclic citrullinated peptide antibodies (anti-CCP Abs), osteoclast activation indicators–the tartrate-resistant acid phosphatase 5b (TRAP5b) and cathepsin K, and bone degradation marker–C-terminal telopeptide of type I collagen (CTX-I) were measured in the bone marrow and peripheral blood of RA patients using ELISAs. We found that ACPAs present in RA bone marrow was associated with increased amounts of TRAP5b, cathepsin K and CTX-I in this location. Levels of IL-8, the key mediator of anti-citrullinated protein antibody (ACPA)-induced bone resorption, were also elevated in bone marrow containing anti-CCP Abs and positively correlated with TRAP5b and cathepsin K concentrations. Higher levels of TRAP5b, cathepsin K, CTX-I and IL-8 in bone marrow compared to peripheral blood indicate local generation of these molecules. Our results complement data from animal studies and highlight the relevance of ACPAs and bone marrow in bone resorption in RA.

Zoledronic Acid Enhanced the Antitumor Effect of Cisplatin on Orthotopic Osteosarcoma by ROS-PI3K/AKT Signaling and Attenuated Osteolysis

Osteoclasts can interact with osteosarcoma to promote the growth of osteosarcoma. Cisplatin is common in adjuvant chemotherapy of osteosarcoma. However, due to chemoresistance, the efficacy is profoundly limited. Previous studies have found that zoledronic acid (ZA) has osteoclast activation inhibition and antitumor effect. However, the combined effect of ZA and cisplatin on osteosarcoma remains unclear. In vitro, the effects of ZA and cisplatin alone or in combination on 143B cell activity, proliferation, apoptosis, and ROS-PI3K/AKT signaling were detected. At the same time, the effect of ZA and cisplatin on osteoclast formation, survival, and activity was detected by TRAP staining and bone plate absorption test. These were further verified in mice. The results showed that in vitro, compared with the single treatment and control, the combination of ZA and cisplatin could significantly inhibit the activity and proliferation of 143B cells and induced their apoptosis and further promoted the generation of ROS and inhibited the phosphorylation of PI3K and AKT. ROS scavenger and the agonist of the PI3K/AKT pathway could reverse these results. In addition, cisplatin in synergy with ZA could significantly inhibit osteoclast formation and survival to reduce bone plate absorption. In vivo, compared with the single group, the tumor volume and cell proliferation were significantly reduced, apoptosis and necrosis of tumor cells increased, and TRAP+ osteoclasts and osteolysis destruction decreased in the combined group. In conclusion, ZA enhanced the antitumor effect of cisplatin on osteosarcoma by ROS-PI3K/AKT signaling, reducing the chemoresistance and osteoclast activation to enhance chemotherapy and inhibit osteolysis. And this present study raised the possibility that combining ZA and cisplatin may represent a novel strategy against osteosarcoma.