Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.     

Piperlongumine Inhibits Titanium Particles-Induced Osteolysis,Osteoclast Formation,and RANKL-Induced Signaling Pathways

Wear particle-induced aseptic loosening is the most common complication of total joint arthroplasty (TJA). Excessive osteoclast formation and bone resorptive activation have been considered to be responsible for extensive bone destruction and prosthesis failure. Therefore, identification of anti-osteoclastogenesis agents is a potential therapy strategy for the treatment of aseptic loosening and other osteoclast-related osteolysis diseases. In the present study, we reported, for the first time, that piperlongumine (PL), a key alkaloid compound from Piper longum fruits, could significantly suppress the formation and activation of osteoclasts. Furthermore, PL effectively decreased the mRNA expressions of osteoclastic marker genes such as tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), and cathepsin K (CTSK). In addition, PL suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced activations of MAPKs (ERK, JNK and p38) and NF-κB, which down-regulated the protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Using a titanium (Ti) particle-induced calvarial osteolysis model, we demonstrated that PL could ameliorate Ti particle-induced bone loss in vivo. These data provide strong evidence that PL has the potential to treat osteoclast-related diseases including periprosthetic osteolysis (PPO) and aseptic loosening.     

Influence of Cu2+ on Osteoclast Formation and Activity In Vitro

Background: Copper-containing biomaterials are increasingly applied for bone regeneration due to their pro-angiogenetic, pro-osteogenetic and antimicrobial properties. Therefore, the effect of Cu²⁺ on osteoclasts, which play a major role in bone remodeling was studied in detail. Methods: Human primary osteoclasts, differentiated from human monocytes were differentiated or cultivated in the presence of Cu²⁺. Osteoclast formation and activity were analyzed by measurement of osteoclast-specific enzyme activities, gene expression analysis and resorption assays. Furthermore, the glutathione levels of the cells were checked to evaluate oxidative stress induced by Cu²⁺. Results: Up to 8 µM Cu²⁺ did not induce cytotoxic effects. Activity of tartrate-resistant acid phosphatase (TRAP) was significantly increased, while other osteoclast specific enzyme activities were not affected. However, gene expression of TRAP was not upregulated. Resorptive activity of osteoclasts towards dentin was not changed in the presence of 8 µM Cu²⁺ but decreased in the presence of extracellular bone matrix. When Cu²⁺ was added to mature osteoclasts TRAP activity was not increased and resorption decreased only moderately. The glutathione level of both differentiating and mature osteoclasts was significantly decreased in the presence of Cu²⁺. Conclusions: Differentiating and mature osteoclasts react differently to Cu²⁺. High TRAP activities are not necessarily related to high resorption.     

Impacts of Hypoxia on Osteoclast Formation and Activity: Systematic Review

Hypoxia is evident in several bone diseases which are characterized by excessive bone resorption by osteoclasts, the bone-resorbing cells. The effects of hypoxia on osteoclast formation and activities are widely studied but remain inconclusive. This systematic review discusses the studies reporting the effect of hypoxia on osteoclast differentiation and activity. A literature search for relevant studies was conducted through SCOPUS and PUBMED MEDLINE search engines. The inclusion criteria were original research articles presenting data demonstrating the effect of hypoxia or low oxygen on osteoclast formation and activity. A total of 286 studies were identified from the search, whereby 20 studies were included in this review, consisting of four in vivo studies and 16 in vitro studies. In total, 12 out of 14 studies reporting the effect of hypoxia on osteoclast activity indicated higher bone resorption under hypoxic conditions, 14 studies reported that hypoxia resulted in more osteoclasts, one study found that the number remained unchanged, and five studies indicated that the number decreased. In summary, examination of the relevant literature suggests differences in findings between studies, hence the impact of hypoxia on osteoclasts remains debatable, even though there is more evidence to suggest it promotes osteoclast differentiation and activity.     

TNF-α Activating Osteoclasts in Patients with Psoriatic Arthritis Enhances the Recruitment of Osteoclast Precursors: A Plausible Role of WNT5A-MCP-1 in Osteoclast Engagement in Psoriatic Arthritis

Psoriatic arthritis (PsA) results from joint destruction by osteoclasts. The promising efficacy of TNF-α blockage indicates its important role in osteoclastogenesis of PsA. WNT ligands actively regulate osteoclastogenesis. We investigated how WNT ligands activate osteoclasts amid the TNF-α milieu in PsA. We first profiled the expression of WNT ligands in CD14⁺ monocyte-derived osteoclasts (MDOC) from five PsA patients and five healthy controls (HC) and then validated the candidate WNT ligands in 32 PsA patients and 16 HC. Through RNA interference against WNT ligands in MDOC, we determined the mechanisms by which TNF-α exerts its effects on osteclastogenesis or chemotaxis. WNT5A was selectively upregulated by TNF-α in MDOC from PsA patients. The number of CD68⁺WNT5A⁺ osteoclasts increased in PsA joints. CXCL1, CXCL16, and MCP-1 was selectively increased in supernatants of MDOC from PsA patients. RNA interference against WNT5A abolished the increased MCP-1 from MDOC and THP-1-cell-derived osteoclasts. The increased migration of osteoclast precursors (OCP) induced by supernatant from PsA MDOC was abolished by the MCP-1 neutralizing antibody. WNT5A and MCP-1 expressions were decreased in MDOC from PsA patients treated by biologics against TNF-α but not IL-17. We conclude that TNF-α recruits OCP by increased MCP-1 production but does not directly activate osteoclastogenesis in PsA.     

Effect of N-Vinyl-2-Pyrrolidone (NVP), a Bromodomain-Binding Small Chemical,on Osteoblast and Osteoclast Differentiation and Its Potential Application for Bone Regeneration

The human skeleton is a dynamic and remarkably organized organ system that provides mechanical support and performs a variety of additional functions. Bone tissue undergoes constant remodeling; an essential process to adapt architecture/resistance to growth and mechanical needs, but also to repair fractures and micro-damages. Despite bone’s ability to heal spontaneously, certain situations require an additional stimulation of bone regeneration, such as non-union fractures or after tumor resection. Among the growth factors used to increase bone regeneration, bone morphogenetic protein-2 (BMP2) is certainly the best described and studied. If clinically used in high quantities, BMP2 is associated with various adverse events, including fibrosis, overshooting bone formation, induction of inflammation and swelling. In previous studies, we have shown that it was possible to reduce BMP2 doses significantly, by increasing the response and sensitivity to it with small molecules called “BMP2 enhancers”. In the present study, we investigated the effect of N-Vinyl-2-pyrrolidone (NVP) on osteoblast and osteoclast differentiation in vitro and guided bone regeneration in vivo. We showed that NVP increases BMP2-induced osteoblast differentiation and decreases RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, in a rabbit calvarial defect model, the histomorphometric analysis revealed that bony bridging and bony regenerated area achieved with NVP-loaded poly (lactic-co-glycolic acid (PLGA) membranes were significantly higher compared to unloaded membranes. Taken together, our results suggest that NVP sensitizes BMP2-dependent pathways, enhances BMP2 effect, and inhibits osteoclast differentiation. Thus, NVP could prove useful as “osteopromotive substance” in situations where a high rate of bone regeneration is required, and in the management of bone diseases associated with excessive bone resorption, like osteoporosis.     

R-Spondin2, a Positive Canonical WNT Signaling Regulator,Controls the Expansion and Differentiation of Distal Lung Epithelial Stem/Progenitor Cells in Mice

The lungs have a remarkable ability to regenerate damaged tissues caused by acute injury. Many lung diseases, especially chronic lung diseases, are associated with a reduced or disrupted regeneration potential of the lungs. Therefore, understanding the underlying mechanisms of the regenerative capacity of the lungs offers the potential to identify novel therapeutic targets for these diseases. R-spondin2, a co-activator of WNT/β-catenin signaling, plays an important role in embryonic murine lung development. However, the role of Rspo2 in adult lung homeostasis and regeneration remains unknown. The aim of this study is to determine Rspo2 function in distal lung stem/progenitor cells and adult lung regeneration. In this study, we found that robust Rspo2 expression was detected in different epithelial cells, including airway club cells and alveolar type 2 (AT2) cells in the adult lungs. However, Rspo2 expression significantly decreased during the first week after naphthalene-induced airway injury and was restored by day 14 post-injury. In ex vivo 3D organoid culture, recombinant RSPO2 promoted the colony formation and differentiation of both club and AT2 cells through the activation of canonical WNT signaling. In contrast, Rspo2 ablation in club and AT2 cells significantly disrupted their expansion capacity in the ex vivo 3D organoid culture. Furthermore, mice lacking Rspo2 showed significant defects in airway regeneration after naphthalene-induced injury. Our results strongly suggest that RSPO2 plays a key role in the adult lung epithelial stem/progenitor cells during homeostasis and regeneration, and therefore, it may be a potential therapeutic target for chronic lung diseases with reduced regenerative capability.     

The Role of Extracellular Vesicles (EVs) in the Epigenetic Regulation of Bone Metabolism and Osteoporosis

Extracellular vesicles (EVs) are complex phospholipidic structures actively released by cells. EVs are recognized as powerful means of intercellular communication since they contain many signaling molecules (including lipids, proteins, and nucleic acids). In parallel, changes in epigenetic processes can lead to changes in gene function and finally lead to disease onset and progression. Recent breakthroughs have revealed the complex roles of non-coding RNAs (microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)) in epigenetic regulation. Moreover, a substantial body of evidence demonstrates that non-coding RNAs can be shuttled among the cells and tissues via EVs, allowing non-coding RNAs to reach distant cells and exert systemic effects. Resident bone cells, including osteoclasts, osteoblasts, osteocytes, and endothelial cells, are tightly regulated by non-coding RNAs, and many of them can be exported from the cells to neighboring ones through EVs, triggering pathological conditions. For these reasons, researchers have also started to exploit EVs as a theranostic tool to address osteoporosis. In this review, we summarize some recent findings regarding the EVs’ involvement in the fine regulation of non-coding RNAs in the context of bone metabolism and osteoporosis.     

The Role of CXCL16 in the Pathogenesis of Cancer and Other Diseases

CXCL16 is a chemotactic cytokine belonging to the α-chemokine subfamily. It plays a significant role in the progression of cancer, as well as the course of atherosclerosis, renal fibrosis, and non-alcoholic fatty liver disease (NAFLD). Since there has been no review paper discussing the importance of this chemokine in various diseases, we have collected all available knowledge about CXCL16 in this review. In the first part of the paper, we discuss background information about CXCL16 and its receptor, CXCR6. Next, we focus on the importance of CXCL16 in a variety of diseases, with an emphasis on cancer. We discuss the role of CXCL16 in tumor cell proliferation, migration, invasion, and metastasis. Next, we describe the role of CXCL16 in the tumor microenvironment, including involvement in angiogenesis, and its significance in tumor-associated cells (cancer associated fibroblasts (CAF), microglia, tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), mesenchymal stem cells (MSC), myeloid suppressor cells (MDSC), and regulatory T cells (T_reg)). Finally, we focus on the antitumor properties of CXCL16, which are mainly caused by natural killer T (NKT) cells. At the end of the article, we summarize the importance of CXCL16 in cancer therapy.     

Using VBIM Technique to Discover ARMC4/ODAD2 as a Novel Negative Regulator of NF-κB and a New Tumor Suppressor in Colorectal Cancer

Since nuclear factor (NF) κB plays pivotal roles in inflammation and cancer, understanding its regulation holds great promise for disease therapy. Using the powerful validation-based insertional mutagenesis (VBIM) technique established by us previously, we discovered armadillo repeat-containing protein 4 (ARMC4)/outer dynein arm docking complex subunit 2 (ODAD2), a rarely studied protein known to date, as a novel negative regulator of NF-κB in colorectal cancer (CRC). High expression of ARMC4 downregulated the expression of NF-κB-dependent genes, dramatically reduced NF-κB activity, cellular proliferation, anchorage-independent growth, and migratory ability in vitro, and significantly decreased xenograft tumor growth in vivo. Co-immunoprecipitation experiments demonstrated that ARMC4 forms a complex with NF-κB. Importantly, the lower ARMC4 expression in patient tumors than normal tissues indicates its potential tumor suppressor function in CRC. Collectively, we uncovered a completely new facet of ARMC4 function by identifying it as a novel NF-κB negative regulator, thus uncovering ARMC4 as a potential new therapeutic target in CRC.     

Bone Formation Ability and Cell Viability Enhancement of MC3T3-E1 Cells by Ferrostatin-1 a Ferroptosis Inhibitor of Cancer Cells

Recently, ferroptosis has gained scientists’ attention as an iron-related regulated necrosis. However, not many reports have investigated the effect of ferroptosis on bone. Therefore, with the present study, we assessed the effect of ferroptosis inhibition using ferrostatin-1 on the MC3T3-E1 pre-osteoblast cell. Cell images, cell viability, alkaline phosphatase activity test, alizarin red staining, and RUNX2 gene expression using real-time PCR were applied to investigate the effects of ferrostatin and erastin on MC3T3-E1 osteoblast cells. Erastin was used as a well-known ferroptosis inducer reagent. Erastin with different concentrations ranging from 0 to 50 µmol/L was used for inducing cell death. The 25 µmol/L erastin led to controllable partial cell death on osteoblast cells. Ferrostatin-1 with 0 to 40 µmol/L was used for cell doping and cell death inhibition effect. Ferrostatin-1 also displayed a recovery effect on the samples, which had already received the partially artificial cell death by erastin. Cell differentiation, alizarin red staining, and RUNX2 gene expression confirmed the promotion of the bone formation ability effect of ferrostatin-1 on osteoblast cells. The objective of this study was to assess ferrostatin-1’s effect on the MC3T3-E1 osteoblast cell line based on its ferroptosis inhibitory property.     

miR2119, a Novel Transcriptional Regulator,Plays a Positive Role in Woody Plant Drought Tolerance by Mediating the Degradation of the CkBI-1 Gene Associated with Apoptosis

Caragana korshinskii, an important vegetation restoration species with economic and ecological benefits in the arid region of northwest China, is characterized by significant drought tolerance. However, the underlying molecular mechanisms by which miRNAs confer this trait in C. korshinskii are unclear. Here, we investigate the effect of CkmiR2119 on drought tolerance and identified its target gene, CkBI-1. A negative correlation of CkmiR2119 and CkBI-1 in both stems and leaves in a drought gradient treatment followed by target gene validation suggest that CkmiR2119 might negatively regulate CkBI-1. Consistently, a decrease in the expression of the CkBI-1 gene was observed after both transient transformation and stable transformation of CkamiR2119 in tobacco (Nicotiana tabacum). Moreover, the physiological analysis of CkamiR2119 and CkBI-1 transgenic plants further indicate that CkmiR2119 can enhance the drought tolerance of C. korshinskii in two aspects: (i) downregulating CkBI-1 expression to accelerate vessel maturation in stems; (ii) contributing to a higher level of CkBI-1 in mesophyll cells to inhibit programmed cell death (PCD). This work reveals that CkmiR2119 can increase plants’ drought tolerance by downregulating the expression of CkBI-1, providing a theoretical basis to improve plants’ ability to withstand stress tolerance by manipulating miRNAs.     

Connecting the Neurobiology of Developmental Brain Injury: Neuronal Arborisation as a Regulator of Dysfunction and Potential Therapeutic Target

Neurodevelopmental disorders can derive from a complex combination of genetic variation and environmental pressures on key developmental processes. Despite this complex aetiology, and the equally complex array of syndromes and conditions diagnosed under the heading of neurodevelopmental disorder, there are parallels in the neuropathology of these conditions that suggest overlapping mechanisms of cellular injury and dysfunction. Neuronal arborisation is a process of dendrite and axon extension that is essential for the connectivity between neurons that underlies normal brain function. Disrupted arborisation and synapse formation are commonly reported in neurodevelopmental disorders. Here, we summarise the evidence for disrupted neuronal arborisation in these conditions, focusing primarily on the cortex and hippocampus. In addition, we explore the developmentally specific mechanisms by which neuronal arborisation is regulated. Finally, we discuss key regulators of neuronal arborisation that could link to neurodevelopmental disease and the potential for pharmacological modification of arborisation and the formation of synaptic connections that may provide therapeutic benefit in the future.