Unique expression and critical role of metallothionein 3 in the control of osteoclastogenesis and osteoporosis.

Bone homeostasis is maintained by an intricate balance between osteoclasts and osteoblasts, which becomes disturbed in osteoporosis. Metallothioneins (MTs) are major contributors in cellular zinc regulation. However, the role of MTs in bone cell regulation has remained unexplored. Single-cell RNA sequencing analysis discovered that, unlike the expression of other MT members, the expression of MT3 was unique to osteoclasts among various macrophage populations and was highly upregulated during osteoclast differentiation. This unique MT3 upregulation was validated experimentally and supported by ATAC sequencing data analyses. Downregulation of MT3 by gene knockdown or knockout resulted in excessive osteoclastogenesis and exacerbated bone loss in ovariectomy-induced osteoporosis. Transcriptome sequencing of MT3 knockdown osteoclasts and gene set enrichment analysis indicated that the oxidative stress and redox pathways were enriched, which was verified by MT3-dependent regulation of reactive oxygen species (ROS). In addition, MT3 deficiency increased the transcriptional activity of SP1 in a manner dependent on intracellular zinc levels. This MT3-zinc-SP1 axis was crucial for the control of osteoclasts, as zinc chelation and SP1 knockdown abrogated the promotion of SP1 activity and osteoclastogenesis by MT3 deletion. Moreover, SP1 bound to the NFATc1 promoter, and overexpression of an inactive SP1 mutant negated the effects of MT3 deletion on NFATc1 and osteoclastogenesis. In conclusion, MT3 plays a pivotal role in controlling osteoclastogenesis and bone metabolism via dual axes involving ROS and SP1. The present study demonstrated that MT3 elevation is a potential therapeutic strategy for osteolytic bone disorders, and it established for the first time that MT3 is a crucial bone mass regulator.

PINK1 restrains periodontitis-induced bone loss by preventing osteoclast mitophagy impairment.

The oral colonization of periodontal pathogens onto gingival tissues establishes hypoxic microenvironment, often disrupting periodontal homeostasis in conjunction with oxidative stress. The association between reactive oxygen species (ROS) and osteolytic periodontitis have been suggested by recent studies. PTEN-induced kinase 1 (PINK1), a mitochondrial serine/threonine kinase, is an essential protein for mitochondrial quality control as it protects cells from oxidative stress by promoting degradation of damaged mitochondria through mitophagy. However, the pathophysiological roles of PINK1 in osteoclast-mediated bone loss have not been explored. Here we aimed to determine whether PINK1 plays a role in the regulation of osteoclastogenesis and alveolar bone resorption associated with periodontitis. C57BL/6 wild type (WT) and Pink1 knockout (KO) mice were subjected to ligature-induced periodontitis (LIP), and alveolar bones were evaluated by µCT-analysis and tartrate-resistant acid phosphatase (TRAP) staining. The µCT-analysis showed that bone volume fraction and travecular thickness were lower in Pink1 KO compared to WT mice. The number of TRAP-positive osteoclasts was markedly increased in the periodontal tissues of Pink1 KO mice with LIP. The genetic silencing or deletion of Pink1 promoted excessive osteoclast differentiation and bone resorption in vitro, as respectively indicated by TRAP staining and resorption pits on dentin slices. PINK1 deficiency led to mitochondrial instabilities as indicated by confocal microscopy of mitochondrial ROS, mitochondrial oxygen consumption rate (OCR) analysis, and transmission electron microscopy (TEM). Consequently, a significant increase in Ca2+-nuclear factor of activated T cells 1 (NFATc1) signaling was also found. On the other hand, restoration of mitophagy and autophagy by spermidine (SPD) treatment and the resolution of oxidative stress by N-acetyl-l-cysteine (NAC) treatment protected PINK1 deficiency-induced excessive generation of osteoclasts. Taken together, our findings demonstrate that PINK1 is essential for maintaining mitochondrial homeostasis during osteoclast differentiation. Therefore, targeting PINK1 may provide a novel therapeutic strategy for severe periodontitis with fulminant osteolysis.

PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway.

Osteoclast generation from monocyte/macrophage lineage precursor cells needs to be tightly regulated to maintain bone homeostasis and is frequently over-activated in inflammatory conditions. PARK2, a protein associated with Parkinson's disease, plays an important role in mitophagy via its ubiquitin ligase function. In this study, we investigated whether PARK2 is involved in osteoclastogenesis. PARK2 expression was found to be increased during the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. PARK2 gene silencing with siRNA significantly reduced osteoclastogenesis induced by RANKL, LPS (lipopolysaccharide), TNFα (tumor necrosis factor α), and IL-1ß (interleukin-1ß). On the other hand, overexpression of PARK2 promoted osteoclastogenesis. This regulation of osteoclastogenesis by PARK2 was mediated by IKK (inhibitory κB kinase) and NF-κB activation while MAPK (mitogen-activated protein kinases) activation was not involved. Additionally, administration of PARK2 siRNA significantly reduced osteoclastogenesis and bone loss in an in vivo model of inflammatory bone erosion. Taken together, this study establishes a novel role for PARK2 as a positive regulator in osteoclast differentiation and inflammatory bone destruction.

Sugar alcohol-based polymeric gene carriers: Synthesis, properties and gene therapy applications.

Advances in the field of nanomedicine have led to the development of various gene carriers with desirable cellular responses. However, unfavorable stability and physicochemical properties have hindered their applications in vivo. Therefore, multifunctional, smart nanocarriers with unique properties to overcome such drawbacks are needed. Among them, sugar alcohol-based nanoparticle with abundant surface chemistry, numerous hydroxyl groups, acceptable biocompatibility and biodegradable property are considered as the recent additions to the growing list of non-viral vectors. In this review, we present some of the major advances in our laboratory in developing sugar-based polymers as non-viral gene delivery vectors to treat various diseases. We also discuss some of the open questions in this field. STATEMENT OF SIGNIFICANCE: Recently, the development of sugar alcohol-based polymers conjugated with polyethylenimine (PEI) has attracted tremendous interest as gene delivery vectors. First, the natural backbone of polymers with their numerous hydroxyl groups display a wide range of hyperosmotic properties and can thereby enhance the cellular uptake of genetic materials via receptor-mediated endocytosis. Second, conjugation of a PEI backbone with sugar alcohols via Michael addition contributes to buffering capacity and thereby the proton sponge effect. Last, sugar alcohol based gene delivery systems improves therapeutic efficacy both in vitro and in vivo.

Biodegradable Polymeric Nanocarrier-Based Immunotherapy in Hepatitis Vaccination.

Various commercial vaccines are used for immunization against hepatitis B. However, these immunotherapeutic vaccines require invasive administration, which can induce side effects, and require multiple shots to elicit an immune response, limiting their efficacy. Compared to traditional hepatitis B vaccines, polymer nanoparticles have more advantageous inherent properties as vaccine delivery carriers, providing increased stability of encapsulated antigen, the possibility of single-shot immunotherapy, and the capability of mucosal administration, which allows various routes of vaccination. In this review, we present up-to-date information on the potential of a biodegradable nanoparticle-based delivery system in treating hepatitis B. We also discuss the application of nanoparticles in various vaccines and highlighted strategies for eliciting an appropriate immune response.

Targeted Delivery of siRNA Therapeutics using Ligand Mediated Biodegradable Polymeric Nanocarriers.

BACKGROUND: Cancer poses a major public health issue, is linked with high mortality rates across the world, and shows a strong interplay between genetic and environmental factors. To date, common therapeutics, including chemotherapy, immunotherapy, and radiotherapy, have made significant contributions to cancer treatment, although diverse obstacles for achieving the permanent "magic bullet" cure have remained. Recently, various anticancer therapeutic agents designed to overcome the limitations of these conventional cancer treatments have received considerable attention. One of these promising and novel agents is the siRNA delivery system; however, poor cellular uptake and altered siRNA stability in physiological environments have limited its use in clinical trials. Therefore, developing the ideal siRNA delivery system with low cytotoxicity, improved siRNA stability in the body's circulation, and prevention of its rapid clearance from bodily fluids, is rapidly emerging as an innovative therapeutic strategy to combat cancer. Moreover, active targeting using ligand moieties which bind to over-expressed receptors on the surface of cancer cells would enhance the therapeutic efficiency of siRNA. CONCLUSION: In this review, we provide 1) an overview of the non-viral carrier associated with siRNA delivery for cancer treatment, and 2) a description of the five major cancer-targeting ligands.

Sugar-based gene delivery systems: Current knowledge and new perspectives.

Carbohydrates, one of the most abundant natural compounds and key participants in many biological processes, are relevant in medical and industrial fields. In comparison with synthetic polymers, carbohydrates are biocompatible and have intrinsic targeting properties, enabling them to interact with cell-surface receptors. Among the different carbohydrates, polysaccharides are naturally occurring biological molecules with tremendous potential for biomedical applications. The physicochemical properties of these polysaccharide based nanoparticles, such as excellent biocompatibility, surface charge to interact with nucleic acids, low toxicity and cost effectiveness make them superior carriers for nanomedicine. In addition to variety of physicochemical properties, polysaccharides allow the great ease of chemical modification which enables the preparation of wide range of nanoparticles. In this review, we present the state-of-the-art information on the potential of polysaccharides-based polymers as non-viral gene delivery vectors in treating various diseases. Then, we discuss the chemical modification and structure/property relationship of carbohydrates.

Muscular involvement of Behçet's disease: ultrasonography, computed tomography, and magnetic resonance imaging findings.

Behçet's disease (BD) is a form of systemic vasculitis with the classic triad of recurrent oral and genital ulcers along with uveitis. In BD, muscular involvement is very rare. We report a case of muscular involvement in BD with characteristic findings using ultrasonography, computed tomography, and magnetic resonance imaging.