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Osteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

Summary One major challenge in realizing cell-based therapy for treating muscle-wasting disorders is the difficulty in obtaining therapeutically meaningful amounts of engraftable cells. We have previously described a method to generate skeletal myogenic progenitors with exceptional engraftability from pluripotent stem cells via teratoma formation. Here, we show that these cells are functionally expandable in vitro while retaining their in vivo regenerative potential. Within 37 days in culture, teratoma-derived skeletal myogenic progenitors were expandable to a billion-fold. Similar to their freshly sorted counterparts, the expanded cells expressed PAX7 and were capable of forming multinucleated myotubes in vitro. Importantly, these cells remained highly regenerative in vivo. Upon transplantation, the expanded cells formed new DYSTROPHIN+ fibers that reconstituted up to 40% of tibialis anterior muscle volume and repopulated the muscle stem cell pool. Our study thereby demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells for transplantation., Graphical abstract, Highlights • Skeletal myogenic progenitors from PSC-derived teratomas are expandable in vitro • Expanded cells are PAX7+ and are capable of differentiating into MHC+ myotubes • Expanded cells engraft and regenerate fibers with adult myosins and innervation • Expanded cells repopulate the muscle stem cell niche after transplantation, In this article, Chan and colleagues show that skeletal myogenic progenitors obtained from PSC-derived teratomas are expandable in cultures and remain highly regenerative. Upon transplantation, the expanded cells robustly engraft to form new muscle fibers and reconstitute the muscle stem cell pool. This study is an advance toward generating large quantities of engraftable skeletal myogenic progenitors for downstream applications.

Screening custom‐made libraries of inhibitors may reveal novel drugs for treating pancreatic cancer. In this manner, we identified ispinesib as a candidate and attempted to determine its clinical efficacy and the biological significance of its functional target Eg5 in pancreatic cancer. One hundred compounds in our library were screened for candidate drugs using cell cytotoxicity assays. Ispinesib was found to mediate effective antitumor effects in pancreatic cancer. The clinical significance of the expression of the ispinesib target Eg5 was investigated in 165 pancreatic cancer patients by immunohistochemical staining and in Eg5‐positive pancreatic cancer patient‐derived xenograft (PDX) mouse models. Patients with Eg5‐positive tumors experienced significantly poorer clinical outcomes than those not expressing Eg5 (overall survival; P, Ispinesib mediated anti–proliferative effects and significantly reduced the growth of Eg5‐positive pancreatic cancer in a patient‐derived xenograft mouse model. Moreover, Eg5‐positivity was associated with poorer clinical outcomes in pancreatic cancer patients. Our results document a clinical indication for ispinesib administration and may contribute to improving the prognosis of pancreatic cancer patients.

Hard palate consists anteriorly of the palatal process of the maxilla (ppmx) and posteriorly of the palatal process of the palatine (ppp). Currently, palatal osteogenesis is receiving increasing attention. This is the first study to provide an overview of the osteogenesis process of the mouse hard palate. We found that the period in which avascular mesenchymal condensation becomes a vascularized bone structure corresponds to embryonic day (E) 14.5 to E16.5 in the hard palate. The ppmx and ppp differ remarkably in morphology and molecular respects during osteogenesis. Osteoclasts in the ppmx and ppp are heterogeneous. There was a multinucleated giant osteoclast on the bone surface at the lateral-nasal side of the ppmx, while osteoclasts in the ppp were more abundant and adjacent to blood vessels but were smaller and had fewer nuclei. In addition, bone remodeling in the hard palate was asymmetric and exclusively occurred on the nasal side of the hard palate at E18.5. During angiogenesis, CD31-positive endothelial cells were initially localized in the surrounding of palatal mesenchymal condensation and then invaded the condensation in a sprouting fashion. At the transcriptome level, we found 78 differentially expressed genes related to osteogenesis and angiogenesis between the ppmx and ppp. Fifty-five related genes were up/downregulated from E14.5 to E16.5. Here, we described the morphogenesis and the heterogeneity in the osteogenic and angiogenic genes profiles of the ppmx and ppp, which are significant for subsequent studies of normal and abnormal subjects.

The objective of this study was to find out if suppression of NF-kB complex function by p65-TMD-linked PTD could reduce host inflammation and bone resorption at peri-implantitis sites in rats. Twenty-one male 5-week-old SD rats were divided into three groups: untreated control group (A), silk-induced peri-implantitis group (B), and nt (nucleus transducible)-p65-TMD-treated, silk-induced peri-implantitis group (C). Implant sulcus of a rat in group C were divided into two groups, namely group Cp and Cb. Palatal implant sulcus where nt-p65-TMD solution was applied with an insulin syringe were assigned to group Cp. Buccal implant sulcus without topical nt-p65-TMD application were assigned to group Cb. H&E staining, TRAP staining, and immunohistological staining were done. The crestal bone levels of group A were significantly higher than those of group B at p

Bone-resorbing osteoclasts (OCLs) are multinucleated phagocytes, whose central roles in regulating bone formation and homeostasis are critical for normal health and development. OCLs are produced from precursor monocytes in a multistage process that includes initial differentiation, cell-cell fusion, and subsequent functional and morphological maturation; the molecular regulation of osteoclastogenesis is not fully understood. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as an essential regulator specifically of OCL maturation. Monocytes from PTPRJ-deficient (JKO) mice differentiate and fuse normally, but their maturation into functional OCLs and their ability to degrade bone are severely inhibited. In agreement, mice lacking PTPRJ throughout their bodies or only in OCLs exhibit increased bone mass due to reduced OCL-mediated bone resorption. We further show that PTPRJ promotes OCL maturation by dephosphorylating the M-CSF receptor (M-CSFR) and Cbl, thus reducing the ubiquitination and degradation of the key osteoclastogenic transcription factor NFATc1. Loss of PTPRJ increases ubiquitination of NFATc1 and reduces its amounts at later stages of osteoclastogenesis, thereby inhibiting OCL maturation. PTPRJ thus fulfills an essential and cell-autonomous role in promoting OCL maturation by balancing between the pro- and anti-osteoclastogenic activities of the M-CSFR and maintaining NFATc1 expression during late osteoclastogenesis.

While the majority of cells contain a single nucleus, cell types such as trophoblasts, osteoclasts, and skeletal myofibers require multinucleation. One advantage of multinucleation can be the assignment of distinct functions to different nuclei, but comprehensive interrogation of transcriptional heterogeneity within multinucleated tissues has been challenging due to the presence of a shared cytoplasm. Here, we utilized single-nucleus RNA-sequencing (snRNA-seq) to determine the extent of transcriptional diversity within multinucleated skeletal myofibers. Nuclei from mouse skeletal muscle were profiled across the lifespan, which revealed the presence of distinct myonuclear populations emerging in postnatal development as well as aging muscle. Our datasets also provided a platform for discovery of genes associated with rare specialized regions of the muscle cell, including markers of the myotendinous junction and functionally validated factors expressed at the neuromuscular junction. These findings reveal that myonuclei within syncytial muscle fibers possess distinct transcriptional profiles that regulate muscle biology., Mammalian skeletal muscle is composed of multinucleated myofibers, containing hundreds of nuclei that coordinate cellular function. Here, the authors show that single-nucleus RNA-sequencing reveals rare and emergent myonuclear populations, and uncovers dynamic transcriptional heterogeneity in development and aging.

Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.

Bone morphogenetic protein (BMP) signaling is well known in bone homeostasis. However, the physiological effects of BMP signaling on mandibles are largely unknown, as the mandible has distinct functions and characteristics from other bones. In this study, we investigated the roles of BMP signaling in bone homeostasis of the mandibles by deleting BMP type I receptor Acvr1 in osteoblast lineage cells with Osterix‐Cre. We found mandibular bone loss in conditional knockout mice at the ages of postnatal day 21 and 42 in an age‐dependent manner. The decreased bone mass was related to compromised osteoblast differentiation together with enhanced osteoclastogenesis, which was secondary to the changes in osteoblasts in vivo. In vitro study revealed that deletion of Acvr1 in the mandibular bone marrow stromal cells (BMSCs) significantly compromised osteoblast differentiation. When wild type bone marrow macrophages were cocultured with BMSCs lacking Acvr1 both directly and indirectly, both proliferation and differentiation of osteoclasts were induced as evidenced by an increase of multinucleated cells, compared with cocultured with control BMSCs. Furthermore, we demonstrated that the increased osteoclastogenesis in vitro was at least partially due to the secretion of soluble receptor activator of nuclear factor‐κB ligand (sRANKL), which is probably the reason for the mandibular bone loss in vivo. Overall, our results proposed that ACVR1 played essential roles in maintaining mandibular bone homeostasis through osteoblast differentiation and osteoblast‐osteoclast communication via sRANKL., ACVR1 promotes osteoblastic differentiation and inhibits osteoclastogenesis through osteoblast‐osteoclast communication via sRANKL. ACVR1 plays an essential role in the maintenance of mandibular bone homeostasis.

Background Axolotls have remarkable organ-level regeneration capability. They can regenerate their limbs, tail, brain, gills, and heart. The liver had been considered to be a regenerative organ in these highly regeneration-competent animals. Therefore, no research had been performed on liver regeneration in urodele amphibians. In the present study, we focused on axolotl liver regeneration and found a unique regeneration mechanism compared with other vertebrates. Results Partial hepatectomy (PH) was performed to assess axolotl liver regeneration. Regeneration was assessed using block-face imaging (CoMBi), histology, cell proliferation, weight gain, and Albumin (Alb) + area. Axolotl liver histology was compared with other vertebrates. Axolotl liver consists of Glisson's capsule, sinusoids, and hepatic cord with no apparent lobule structures. Hepatocytes were mononucleated or multinucleated. PH increased the multinucleated hepatocytes and the Alb + area, but there was no apparent liver shape recovery even 40 days after PH. Gene expression pattern suggests that no epimorphic regeneration takes place. We also found that the increase in the number of proliferating hepatocytes was regulated by ERK-signaling. Conclusion Our findings suggest that axolotls, which have epimorphic regeneration ability, regenerate their liver via unique mechanisms, compensatory congestion.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa &beta, (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Osteoporosis (OP) is an age-related osteolytic disease and characterized by low bone mass and more prone to fracture due to active osteoclasts. Proliferating cell nuclear antigen (PCNA) has been long identified as a nuclear protein playing critical roles in the regulation of DNA replication and repair. Recently, a few studies have demonstrated the cytoplasmic localization of PCNA and its function associated with apoptosis in neutrophil and neuroblastoma cells. However, the involvement of PCNA, including the cytoplasmic PCNA, in the osteoclast differentiation remains unclear. In the present study, we show that PCNA is translocated from nucleus to cytoplasm during the RANKL-induced osteoclast differentiation, and localized in the actin belt of mature osteoclast. Knockdown of PCNA significantly affected the integrity of actin belt, the formation of multinucleated osteoclasts, the expression of osteoclast-specific genes, and the in vitro bone resorption. Interactomic study has revealed β-actin as the major interacting partner of the cytoplasmic PCNA, suggesting that cytoplasmic PCNA might play a critical role in the differentiation of osteoclast through regulation of actin-cytoskeleton remodeling. Taken together, our results demonstrate the critical role of cytoplasmic PCNA during the process of osteoclast differentiation, and provided a potential therapeutic target for treatment of osteoclast-related bone diseases.

N16 is an active protein existing in Pinctada martensi. Our previous studies have demonstrated that N16 inhibited osteoclast differentiation in vitro. To better understand how N16 regulates osteoclast differentiation, RAW264.7 cells, a murine monocytic cell line and murine bone marrow-derived macrophages (BMMs) were adopted. Treatment of RAW264.7 cells with RANKL activated osteoclastogenesis and N16 inhibited the formation of multinucleated osteoclasts and TRAP activity. The suppression occurred at the early stage of osteoclastogenesis. Moreover, we found that N16 inhibited PU.1 and MITF expressions, mirroring the inhibition of RANK expressions, indicating that N16 inhibited RANK expression by down-regulating the expressions of MITF and PU.1, thus preventing osteoclastogenesis.

Conditioned medium (CM) (cell secretome) is a cocktail of growth factors, cytokines, and other soluble mediators secreted by cells into a culture medium. These growth factors are fundamental in many cellular processes such as cell growth, differentiation, and others and the composition of these factors is individual for each cell type. Osteoclasts are large multinucleated cells that are responsible for bone resorption. Immune and cancer cells are known to produce different growth factors, which are able to induce or inhibit osteoclast differentiation. Herein, we evaluated the effect of CM obtained from the supernatant of activated and non-activated Jukart-E6 cells, as well as from one murine (B16-F10) and one human melanoma cell line (SK-MEL-28). To induce osteoclast differentiation, murine bone marrow mononuclear cells were cultured in the presence and absence of differentiation factors (DF), such as macrophage colony-stimulating factor, prostaglandin E2, receptor activator of nuclear factor-κB ligand, and CM. We measured the concentration of interleukin 6, tumor necrosis factor-α and interferon γ (IFN-γ) in CM that can inhibit or induce osteoclastogenesis. Our study demonstrated that CM obtained from each cell line suppresses or inhibits osteoclasts formation at early and intermediate stages of differentiation in the absence or presence of DF. CM obtained from activated Jurkat-E6 cells demonstrates a stronger effect when compared with CM from naïve Jurkat-E6 cells or human and murine melanoma cells. Moreover, CM obtained from activated Jurkat-E6 cells shows higher secretion of IFN-γ, which is an inhibitor of osteoclastogenesis, in comparison with CM obtained from the three other cell lines. On the other hand, CM derived from B16-F10 cells showed a smaller inhibitory effect when compared with CM derived from the other cells.

The total flavonoids from Tridax procumbens (TPFs) have been reported significantly to suppress on RANKL‐induced osteoclast differentiation and bone resorption in mouse primary cultured osteoclasts. However, the effects of ethyl ether fraction of Tridax procumbens flavonoids (TPF) on osteoclastogenesis remain unknown. In this study, we investigated the effects of TPF on lipopolysaccharides (LPS)‐induced osteoclast differentiation, actin ring formation, and explored its molecular mechanism in vitro. Matured osteoclast was counted as the number of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells, and activity of osteoclast was assessed by performing the pit formation assays. Real‐time polymerase chain reaction (RT‐PCR) was performed for evaluation of the expression of osteoclast differentiation‐related genes. TPF reduced the TRAP‐positive multinucleated osteoclasts, inhibited TRAP and acid phosphatase (ACP) activities and decreased the expression of osteoclast differentiating genes, including cathepsin K, metalloproteinase‐2 (MMP‐2), MMP‐9, MMP‐13 and osteoclast‐associated receptor (OSCAR). Furthermore, osteoclast‐dependent actin rings formation and resorption pits were dramatically inhibited by the treatment with TPF. TPF markedly decreased the expression levels of transcription factors such as c‐Fos, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and activator protein‐1 (AP‐1). Taken together, our findings indicated that TPF suppressed both osteoclast differentiation and activities. Therefore, TPF might be a promising and emerging drug candidate for the treatment of bone diseases such as osteoporosis.

The role of retinoid-related orphan receptor, one of the transcription factors reported in testis, in testicular function is unclear, so this study was performed to evaluate the qualitative and quantitative changes in the testicular structure of RORα-deficient mice using light-, electron-microscopy, and immunohistochemistry. Among the most striking alterations observed in the testis of the mutant mice were hypospermatogenesis, marked reduction in volume proportions of interstitial tissues and number of Leydig cells, significant decrease in the diameter of seminiferous tubules and height of their epithelium, vacuolation in the epithelium of the seminiferous tubules with occurrence of mast cells, appearance of delay spermiation signs, and changes in sperm morphology. Moreover, the testis of mutant mice showed symplasts, in addition to appearance of multinucleated giant bromophenol-positive cells. ATPase activity was limited to spermatogonia and some primary spermatocytes, with higher alkaline phosphatase expression. Stronger vimentin reaction was immunolocalized to spermatogonia, spermatids, Leydig cells, and Sertoli cells. The expression of CD117 (C-kit, stem cell growth factor receptor) was limited to spermatogonia, primary spermatocytes, and Leydig cells. Seminiferous tubules showed overexpression of vascular endothelial growth factor (VEGF). Transmission electron microscopy examination of the mutant mice revealed abnormal Sertoli cells, hypertrophied spermatogonia, spermatocytes with degenerated mitochondria, and incompletely developed sperms. In conclusion, RORα is one of the essential proteins that regulate testicular structure.

Summary Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021)., Graphical abstract, Highlights • snRNA-seq captures essentially all cell populations present in the skeletal muscle tissue • Can identify rare myonuclei populations, like neuromuscular and myotendinous myonuclei • snRNA-seq circumvents cell-dissociation-induced modifications on gene expression • In contrast to single-cell RNA-seq, snRNA-seq allows us to process snap frozen biopsies, Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications.

Skeletal muscle plays essential roles in motor function, energy, and glucose metabolism. Skeletal muscle formation occurs through a process called myogenesis, in which a crucial step is the fusion of mononucleated myoblasts to form multinucleated myofibers. The myoblast/myocyte fusion is triggered and coordinated in a muscle-specific way that is essential for muscle development and post-natal muscle regeneration. Many molecules and proteins have been found and demonstrated to have the capacity to regulate the fusion of myoblast/myocytes. Interestingly, two newly discovered muscle-specific membrane proteins, Myomaker and Myomixer (also called Myomerger and Minion), have been identified as fusogenic regulators in vertebrates. Both Myomaker and Myomixer-Myomerger-Minion have the capacity to directly control the myogenic fusion process. Here, we review and discuss the latest studies related to these two proteins, including the discovery, structure, expression pattern, functions, and regulation of Myomaker and Myomixer-Myomerger-Minion. We also emphasize and discuss the interaction between Myomaker and Myomixer-Myomerger-Minion, as well as their cooperative regulatory roles in cell-cell fusion. Moreover, we highlight the areas for exploration of Myomaker and Myomixer-Myomerger-Minion in future studies and consider their potential application to control cell fusion for cell-therapy purposes.

The adhesion morphology of a cell monolayer results in a mechanical force inside cells, between cells, or between cells and substrates. The mechanical force regulates the differentiation of stem cells, but its influence on cell fusion is seldom studied. The present study is focused on osteoclast precursors, RAW264.7 monocytes, which can fuse into multinucleated cells (MNCs) responsible for bone resorption. Cells were cultured on circular and ring-like patterned substrates. Then, cell fusion, cell-substrate traction force, and force-sensitive molecules in different regions were measured and analyzed. Results showed that MNCs mainly appeared in the interior of the ring-like pattern and the central zone of the circular pattern, where both cell-substrate traction force and in-plane maximal shear stress were smaller than that at the patterns' edge. The immunostaining results revealed that F-actin, vinculin, β-catenin, and E-cadherin were highly distributed at the edge of patterns. High seeding density of cells promoted mechanical force-dependent fusion. When calcium-dependent cell-cell connections were inhibited by E-cadherin antibody or low-calcium medium, the fusion into MNCs was greatly reduced. Thus, the morphology of cell monolayer decides the mechanical state of cell-substrate interaction and cell-cell connection, ultimately regulating the fusion of osteoclast precursors.

The receptor activator of nuclear factor-kappa B ligand (RANKL) induces osteoclastogenesis by induction of Ca2+ oscillation, calcineurin activation and translocation into the nucleus of nuclear factor of activated T cells type c1 (NFATc1). Homer proteins are scaffold proteins. They regulate Ca2+ signaling by modulating the activity of multiple Ca2+ signaling proteins. Homers 2 and 3, but not Homer1, also independently affect the interaction between NFATc1 and calcineurin. However, to date, whether and how the Homers are involved in osteoclastogenesis remains unknown. In the present study, we investigated Homer2 and Homer3 roles in Ca2+ signaling and NFATc1 function during osteoclast differentiation. Deletion of Homer2/Homer3 (Homer2/3) markedly decreased the bone density of the tibia, resulting in bone erosion. RANKL-induced osteoclast differentiation is greatly facilitated in Homer2/3 DKO bone marrow-derived monocytes/macrophages (BMMs) due to increased NFATc1 expression and nuclear translocation. However, these findings did not alter RANKL-induced Ca2+ oscillations. Of note, RANKL treatment inhibited Homer proteins interaction with NFATc1, but it was restored by cyclosporine A treatment to inhibit calcineurin. Finally, RANKL treatment of Homer2/3 DKO BMMs significantly increased the formation of multinucleated cells. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation. Specifically, we found that Homer2 and Homer3 regulate NFATc1 function through its interaction with calcineurin to regulate RANKL-induced osteoclastogenesis and bone metabolism.

Prenatal and early life exposure of chlorpyrifos (CPF), a widely used pesticide, is known to cause neuronal deficits and Parkinson's disease (PD). However, data about the effect of its exposure at adult stages on PD-like symptoms and associated bone loss is scanty. In the present study, we investigated the impact of CPF on the behavioral alterations seen in PD using adult Swiss albino mice. PD is often associated with bone loss. Hence, skeletal changes were also evaluated using micro-computed tomography and histology. MPTP was used as a positive control. Cell culture studies using MC3T3E-1, SHSY5Y, and primary osteoclast cultures were done to understand the cellular mechanism for the behavioral and skeletal changes. Our results showed that CPF treatment leads to PD-like symptoms due to the loss of dopaminergic neurons. Moreover, CPF has a deleterious effect on the trabecular bone through both indirect changes in circulating factors and direct stimulation of multinucleate osteoclast cell formation. The impact on the bone mass was even stronger than MPTP. In conclusion, this is the first report demonstrating that CPF induces parkinsonian features in adult Swiss albino mice and it is accompanied by loss of trabecular bone.

Considerable effort has gone into investigating mechanisms that underlie the developmental transition in which mammalian cardiomyocytes (CMs) switch from being able to proliferate during development, to essentially having lost that ability at maturity. This problem is interesting not only for scientific curiosity, but also for its clinical relevance because controlling the ability of mature CMs to replicate would provide a much-needed approach for restoring cardiac function in damaged hearts. In this review, we focus on the propensity of mature mammalian CMs to be multinucleated and polyploid, and the extent to which this may be necessary for normal physiology yet possibly disadvantageous in some circumstances. In this context, we explore whether the concept of the myonuclear domain (MND) in multinucleated skeletal muscle fibers might apply to cardiomyocytes, and whether cardio-MND size might be related to the transition of CMs to become multinuclear. Nuclei in CMs are almost certainly integrators of not only biochemical, but also—because of their central location within the myofibrils—mechanical information, and this multimodal, integrative function in adult CMs—involving molecules that have been extensively studied along with newly identified possibilities—could influence both gene expression as well as replication of the genome and the nuclei themselves.

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

Objective Bone remodeling is mediated by the interaction between osteoblasts and osteoclasts, so does osteoinduction triggered by calcium phosphate (CaP) biomaterials. This study aims to investigate the role and function of osteoclasts in ectopic bone formation induced by CaP biomaterials. Methods Four kinds of mice, two outbred mouse strains (KM and ICR) and two inbred mouse strains (C57BL/6 and BALB/c), were chosen for the experiments. The hydroxyapatite/𝛽-tricalcium phosphate (HA/𝛽-TCP) biomaterials were implanted into the bilateral thigh muscle of each mouse, and then all mice ran on the treadmill to accelerate the ectopic bone formation. Five and ten weeks later, five mice in each group were euthanized and the samples were harvested for electron microscope scanning or histological identification: hematoxylin and eosin (HE), Masson-trichrome and tartrate-resistant acid phosphatase (TRAP) staining, respectively. The inflammation indexes, angiogenesis, and osteogenic ability were compared among the four kinds of mice, and the role of osteoclasts was analyzed based on this evidence. Results The number of multinucleated cells, the number of new blood vessels, and the area percentage of new bone tissues were higher in outbred mouse strains than in inbred mouse strains; and there were more TRAP-positive cells in the outbred mouse strains group. We believe that the monocytes from the peripheral blood could migrate into new bone tissues to form osteoclasts. Conclusion Bone induction could be triggered by CaP biomaterials in mice, and osteoclasts could maintain the dynamic balance between bone resorption and remodeling, and induce the production of new bone marrow tissues.

To investigate the expression of C-JNK, RANKL and OPG after SP600125 administration in cultured dental follicle cells (DFCs). TRAP staining and electron microscope were carried out on day 7 and 9 after coculture of BMMs and DFCs with a ratio of 5:1 in different groups. To determine the effects of SP600125 on the expression of C-JNK, RANKL and OPG mRNA and protein, cultured DFCs were divided into control group, DMSO group and SP600125 groups. Real-time PCR and Western blot analysis were performed to investigate the expression of the mRNA and protein, respectively. TRAP assay indicated that the number of multinucleated osteoclasts in the SP600125 group showed significant decrease compared with that of control (P

Osteoclasts are multinucleated cells responsible for bone resorption. The differentiation of osteoclasts from bone marrow macrophages (BMMs) is induced by receptor activator of NF-κB ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor of RANKL, inhibits osteoclastogenesis by blocking RANKL signaling. Here we investigated the degradation of OPG in vitro. Osteoclasts, but not BMMs, secreted OPG-degrading enzymes. Using mass spectrometry and RNA-sequencing analysis, we identified high-temperature requirement A serine peptidase 1 (HtrA1) as an OPG-degrading enzyme. HtrA1 did not degrade OPG pre-reduced by dithiothreitol, suggesting that HtrA1 recognizes the three-dimensional structure of OPG. HtrA1 initially cleaved the amide bond between leucine 90 and glutamine 91 of OPG, then degraded OPG into small fragments. Inhibitory activity of OPG on RANKL-induced osteoclastogenesis was suppressed by adding HtrA1 in RAW 264.7 cell cultures. These results suggest that osteoclasts potentially prepare a microenvironment suitable for osteoclastogenesis. HtrA1 may be a novel drug target for osteoporosis., Nagahiro Ochiai et al. report that osteoclasts, but not bone marrow macrophages, secrete a serine protease HtrA1 that can degrade osteoprotegerin in the bone microenvironment. Their results suggest HtrA1 recognizes the three-dimensional structure of osteoprotegerin and may function to prepare the microenvironment for osteoclastogenesis.

Previous studies have reported that octacalcium phosphate (OCP) enhances osteoblast differentiation and osteoclast formation during the hydrolysis process to hydroxyapatite (HA). However, the crystal phases that affect the crosstalk between osteoclasts and osteoblasts are unknown, which should determine the bone substitute material's property of OCP. The present study was designed to investigate whether the chemical composition and crystal structure of calcium phosphates affect osteoclast formation and the osteoclast-osteoblast crosstalk. Biodegradable β-tricalcium phosphate (β-TCP) was used as the control material. Osteoclasts were cultured on HA/OCP or HA/TCP disks and their cellular responses were assessed. Both OCP and β-TCP had a similar ability to create multinucleated osteoclasts. However, OCP promoted the expression of complement component 3a (C3a), a positive coupling factor, in osteoclasts, whereas β-TCP enhanced that of EphrinB2 (EfnB2) and collagen triple helix repeat containing 1 (Cthrc1). During osteoclast culture, phosphate ions were released from the crystals, and OCP-HA conversion was advanced in HA/OCP mixtures and OCP. X-ray diffraction analysis revealed no remarkable changes in the crystal structures of HA/TCP mixtures and β-TCP before and after osteoclast culture. These results indicate that the distinct chemical environment induced by the calcium phosphate phases affects the crosstalk between osteoclasts and osteoblasts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1001-1013, 2019.

Rosen et al. identify a role for the centrosomal protein Bsg25D/Ninein in nuclear positioning and microtubule organization in Drosophila muscle fibers. Genetic, cell biological, and atomic force microscopy analyses demonstrate that complex interactions between Bsg25D and the microtubule-associated protein Ensconsin govern myonuclear positioning in Drosophila., Skeletal muscle consists of multinucleated cells in which the myonuclei are evenly spaced throughout the cell. In Drosophila, this pattern is established in embryonic myotubes, where myonuclei move via microtubules (MTs) and the MT-associated protein Ensconsin (Ens)/MAP7, to achieve their distribution. Ens regulates multiple aspects of MT biology, but little is known about how Ens itself is regulated. We find that Ens physically interacts and colocalizes with Bsg25D, the Drosophila homologue of the centrosomal protein Ninein. Bsg25D loss enhances myonuclear positioning defects in embryos sensitized by partial Ens loss. Bsg25D overexpression causes severe positioning defects in immature myotubes and fully differentiated myofibers, where it forms ectopic MT organizing centers, disrupts perinuclear MT arrays, reduces muscle stiffness, and decreases larval crawling velocity. These studies define a novel relationship between Ens and Bsg25D. At endogenous levels, Bsg25D positively regulates Ens activity during myonuclear positioning, but excess Bsg25D disrupts Ens localization and MT organization, with disastrous consequences for myonuclear positioning and muscle function.

Established RAW264.7 cell lines for osteoclastic differentiation has been widely engaged in bone homeostasis research, however, the efficacy of RANKL independently stimulating has rarely been defined, because protocols were usually developed and modified by various laboratories. Otherwise, problematic issues are also lie in the cell's seeding density, RANKL stimulating time point, and distinguishing osteoclastogenesis ability of RANKL-treated RAW264.7 cells. Therefore, in the current study, we examined the efficacy of various concentrations of RANKL-treated RAW264.7 for its osteoclastic differentiation with or without pretreated other costimulators such as: LPS and/or M-CSF. The oteoclastogenesis ability of RANKL-treated RAW264.7 cells was demonstrated by bone resorption pit, F-actin, and osteoclastogenesis specific marker studies. Besides that, through tartrate-resistant acid phosphatase (TRAP) staining, we clarified to start the treatment with 30 ng/ml RANKL at 12 hr after seeded RAW264.7 with the density of 6.25 × 10 3 cells/cm 2 manifested an significantly increased number of multinucleated osteoclastic cells. Overall, our results establishing an optimal method for RANKL independently inducing RAW 264.7 cell osteoclastic differentiation, which could efficiently generate osteoclasts in vitro for significant advances in our understanding of bone biology.

Background The regulatory mechanisms of hematopoiesis and cellular immunity show a high degree of similarity between insects and mammals, and Drosophila has become a good model for investigating cellular immune responses. Jumeau (Jumu) is a member of the winged-helix/forkhead (FKH) transcription factor family and is required for Drosophila development. Adult jumu mutant flies show defective hemocyte phagocytosis and a weaker defense capability against pathogen infection. Here, we further investigated the role of jumu in the regulation of larval hemocyte development and phagocytosis. Methods In vivo phagocytosis assays, immunohistochemistry, Real-time quantitative PCR and immunoblotting were performed to investigate the effect of Jumu on hemocyte phagocytosis. 5-Bromo-2-deoxyUridine (BrdU) labeling, phospho-histone H3 (PH3) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were performed to analyze the proliferation and apoptosis of hemocyte; immunohistochemistry and Mosaic analysis with a repressible cell marker (MARCM) clone analysis were performed to investigate the role of Jumu in the activation of Toll pathway. Results Jumu indirectly controls hemocyte phagocytosis by regulating the expression of NimC1 and cytoskeleton reorganization. The loss of jumu also causes abnormal proliferation and differentiation in circulating hemocytes. Our results suggest that a severe deficiency of jumu leads to the generation of enlarged multinucleate hemocytes by affecting the normal cell mitosis process and induces numerous lamellocytes by activating the Toll pathway. Conclusions Jumu regulates circulating hemocyte differentiation and phagocytosis in Drosophila. Our findings provide new insight into the mechanistic roles of cytoskeleton regulatory proteins in phagocytosis and establish a basis for further analyses of the regulatory mechanism of the mammalian ortholog of Jumu in mammalian innate immunity. Electronic supplementary material The online version of this article (10.1186/s12964-018-0305-3) contains supplementary material, which is available to authorized users.

The Sonic Hedgehog signaling (Shh) pathway has been implicated in both proliferation of myoblast cells and terminal differentiation of muscle fibers, and contradictory results of these effects have been described. To clarify the role of Shh during myogenesis, we decided to study the effects of recombinant Shh and the distribution of Gli-1 during in vitro and in situ embryonic chick skeletal muscle differentiation at later stages of development. Gli-1 was found in small aggregates near the nucleus in mononucleated myoblasts and in multinucleated myotubes both in vitro and in situ chick muscle cells. Some Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle fibers in situ. Recombinant Shh added to in vitro grown muscle cells induced the nuclear translocation of Gli-1, as well as an increase in the number of myoblasts and in the number of nuclei within myotubes. We suggest that Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the rapid cellular redistribution of Gli-1 upon specific signals during muscle differentiation.

Inclusion bodies (IBs) and multinucleate cells can be associated with viral infections; however, IBs and multinucleate cells have been described in normal tissue and with non-viral disease processes in multiple species. We examined fundic stomach from 50 callitrichids histologically for bi- and multinucleate parietal cells and cytoplasmic IBs in gastric epithelial cells. Callitrichids represented included 6 genera: Saguinus (4 spp.), Leontopithecus (1 sp.), Mico (3 spp.), Cebuella (1 sp.), Callithrix (1 sp.), Callimico (1 sp.), and 13 unspecified marmosets. Gastric epithelial IBs were present in 46 of 47 (98%) of the callitrichids from which the stomach was sufficiently well preserved to identify IBs. Cytoplasmic IBs were identified in gastric surface pit epithelial cells (43 of 44, 98%), mucous neck cells (43 of 44, 98%), parietal cells (43 of 44, 98%), and chief cells (43 of 44, 98%). The IBs were eosinophilic, ovoid, round, elongate, or variably indented, sometimes slightly refractile, and 1–6 × 1–13 µm. IBs were sometimes perinuclear and molded around the nucleus. Electron microscopy of the gastric epithelium of one marmoset indicated that IBs were composed of intermediate filaments. The IBs did not stain with immunohistochemical markers for cytokeratin AE1/AE3 or vimentin. Binucleate parietal cells were found in 49 of 50 (98%) callitrichids, and multinucleate parietal cells were observed in 40 of 49 (82%) callitrichids. Gastric epithelial cytoplasmic IBs and bi- and multinucleate parietal cells are likely a normal finding in callitrichids, and, to our knowledge, have not been reported previously.

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell–cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Regulating the number of progeny generated by replicative cell cycles is critical for any organism to best adapt to its environment. Classically, the decision whether to divide further is made after cell division is completed by cytokinesis and can be triggered by intrinsic or extrinsic factors. Contrarily, cell cycles of some species, such as the malaria-causing parasites, go through multinucleated cell stages. Hence, their number of progeny is determined prior to the completion of cell division. This should fundamentally affect how the process is regulated and raises questions about advantages and challenges of multinucleation in eukaryotes. Throughout their life cycle Plasmodium spp. parasites undergo four phases of extensive proliferation, which differ over three orders of magnitude in the amount of daughter cells that are produced by a single progenitor. Even during the asexual blood stage proliferation parasites can produce very variable numbers of progeny within one replicative cycle. Here, we review the few factors that have been shown to affect those numbers. We further provide a comparative quantification of merozoite numbers in several P. knowlesi and P. falciparum parasite strains, and we discuss the general processes that may regulate progeny number in the context of host-parasite interactions. Finally, we provide a perspective of the critical knowledge gaps hindering our understanding of the molecular mechanisms underlying this exciting and atypical mode of parasite multiplication.

To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1(fl/fl)-LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.

The present study was designed to test the hypothesis that osteoclasts appear after or at the same time as the initiation of bone mineralization in developing intramembranous bones. We examined mineral deposition via Von Kossa staining to determine when bone mineralization begins, tartrate-resistant acid phosphatase (TRAP) activity and cathepsin K immunoreactivity to identify the presence of osteoclasts, and their mRNA expression levels to assess osteoclastic differentiation in the embryonic mouse mandible. Cathepsin K-immunopositive cells were detected around the same time as the onset of bone mineralization, whereas TRAP-positive cells appeared prior to bone mineralization. Cathepsin K protein was expressed only in multinucleated osteoclasts, whereas TRAP activity was identified in both mono- and multinucleated cells. During bone development, TRAP-positive cells altered their morphology, which was related to the number of their nuclei. The elevated mRNA levels of TRAP and cathepsin K were consistent with the increased percentage of multinucleated osteoclasts and the progression of bone development. Our study revealed that TRAP-positive cells appear prior to bone mineralization, and TRAP- and cathepsin K-positive multinucleated osteoclasts appear at the same time as the initiation of bone mineralization in embryonic mouse mandibles, suggesting that osteoclasts contribute to bone matrix maturation during intramembranous ossification.

Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell–cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFβ) pathway is active in adult muscle cells throughout fusion. We find TGFβ signaling reduces cell fusion, regardless of the cells’ ability to move and establish cell-cell contacts. In contrast, inhibition of TGFβ signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFβ protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFβR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFβ controls the expression of actin-related genes to reduce cell spreading. TGFβ signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size., The fusion of muscle progenitor cells to form syncytial myofibers is required for skeletal muscle development and regeneration. Here, the authors describe a novel and specific molecular regulation of muscle cell fusion driven by transforming growth factor beta (TGFβ) signaling.

During pregnancy, the appropriate allocation of nutrients between the mother and the fetus is dominated by maternal–fetal interactions, which is primarily governed by the placenta. The syncytiotrophoblast (STB) lining at the outer surface of the placental villi is directly bathed in maternal blood and controls feto–maternal exchange. The STB is the largest multinucleated cell type in the human body, and is formed through syncytialization of the mononucleated cytotrophoblast. However, the physiological advantage of forming such an extensively multinucleated cellular structure remains poorly understood. Here, we discover that the STB uniquely adapts to nutrient stress by inducing the macropinocytosis machinery through repression of mammalian target of rapamycin (mTOR) signaling. In primary human trophoblasts and in trophoblast cell lines, differentiation toward a syncytium triggers macropinocytosis, which is greatly enhanced during amino acid shortage, induced by inhibiting mTOR signaling. Moreover, inhibiting mTOR in pregnant mice markedly stimulates macropinocytosis in the syncytium. Blocking macropinocytosis worsens the phenotypes of fetal growth restriction caused by mTOR-inhibition. Consistently, placentas derived from fetal growth restriction patients display: 1) Repressed mTOR signaling, 2) increased syncytialization, and 3) enhanced macropinocytosis. Together, our findings suggest that the unique ability of STB to undergo macropinocytosis serves as an essential adaptation to the cellular nutrient status, and support fetal survival and growth under nutrient deprivation.

Funder: Swedish Dental Society, Numerous studies have reported on the positive effects of silicon (Si) on bone metabolism, particularly on the stimulatory effects of Si on osteoblast cells and on bone formation. Inhibitory effects of Si on osteoclast formation and bone resorption have also been demonstrated in vitro and are suggested to be mediated indirectly via stromal and osteoblast cells. Direct effects of Si on osteoclasts have been less studied and mostly using soluble Si, but no characterisation of the Si treatment solutions are provided. The aims of the present study were to (a) further investigate the direct inhibitory effects of Si on osteoclastogenesis in RANKL-stimulated RAW264.7 cells, (b) determine at what stage during osteoclastogenesis Si acts upon, and (c) determine if these effects can be attributed to the biologically relevant soluble orthosilicic acid specie. Our results demonstrate that silicon, at 50 μg/ml (or 1.8 mM), does not affect cell viability but directly inhibits the formation of TRAP+ multinucleated cells and the expression of osteoclast phenotypic genes in RAW264.7 cells. The inhibitory effect of Si was clearly associated with the early stages (first 24 hr) of osteoclastogenesis. Moreover, these effects can be attributed to the soluble orthosilicic acid specie.

A 13-year-old castrated male Maine coon cat with a 5-year history of atypical mycobacteriosis was euthanized and submitted for necropsy. The cat had been kept in clinical remission since diagnosis using a combination of the antimycobacterial drug clofazimine and additional multimodal antimicrobial therapy. Grossly, tissues were diffusely discolored red-brown to yellow. Histologically, the myocardial interstitum was expanded by numerous, often multinucleated cells, which were distended by uniformly shaped acicular cytoplasmic spaces. These cells were immunopositive for CD18 and immunonegative for desmin, suggesting a histiocytic rather than muscular origin. Macrophages in other tissues contained similar acicular spaces. Ultrastructurally, the spaces were surrounded by 2 lipid membranes, resembling an autophagosome. Based on the clinical history and histologic, immunohistochemical, and ultrastructural data, we diagnosed clofazimine crystal storage. To our knowledge, this is the first report of clofazimine storage in a cat or within myocardial interstitial macrophages.

Classical Hodgkin lymphoma (cHL) is unique among lymphoid malignancies in several key biological features. (i) The Hodgkin and Reed-Sternberg (HRS) tumor cells are rare among an extensive and complex microenvironment. (ii) They derive from B cells, but have largely lost the B-cell typical gene expression program. (iii) Their specific origin appears to be pre-apoptotic germinal center (GC) B cells. (iv) They consistently develop bi- or multinucleated Reed-Sternberg cells from mononuclear Hodgkin cells. (v) They show constitutive activation of numerous signaling pathways. Recent studies have begun to uncover the basis of these specific features of cHL: HRS cells actively orchestrate their complex microenvironment and attract many distinct subsets of immune cells into the affected tissues, to support their survival and proliferation, and to create an immunosuppressive environment. Reed-Sternberg cells are generated by incomplete cytokinesis and refusion of Hodgkin cells. Epstein-Barr virus (EBV) plays a major role in the rescue of crippled GC B cells from apoptosis and hence is a main player in early steps of lymphomagenesis of EBV+ cHL cases. The analysis of the landscape of genetic lesions in HRS cells so far did not reveal any highly recurrent HRS cell-specific lesions, but major roles of genetic lesions in members of the NF-κB and JAK/STAT pathways and of factors of immune evasion. It is perhaps the combination of the genetic lesions and the peculiar cellular origin of HRS cells that are disease defining. A combination of such genetic lesions and multiple cellular interactions with cells in the microenvironment causes the constitutive activation of many signaling pathways, often interacting in complex fashions. In nodular lymphocyte predominant Hodgkin lymphoma, the GC B cell-derived tumor cells have largely retained their typical GC B-cell expression program and follicular microenvironment. For IgD-positive cases, bacterial antigen triggering has recently been implicated in early stages of its pathogenesis.

Herein we analyze two special routes of the multinucleated cells' formation - the fusion of mononuclear cells and the formation of cell-in-cell structures - in the healthy tissues and in tumorigenesis. There are many theories of tumorigenesis based on the phenomenon of emergence of the hybrid cancer cells. We consider the phenomena, which are rarely mentioned in those theories: namely, cellularization of syncytium or coenocytes, and the reversible or irreversible somatogamy. The latter includes the short-term and the long-term vegetative (somatic) cells' fusions in the life cycles of unicellular organisms. The somatogamy and multinuclearity have repeatedly and independently emerged in various groups of unicellular eukaryotes. These phenomena are among dominant survival and biodiversity sustaining strategies in protists and we admit that they can likely play an analogous role in cancer cells.

Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL–driven bone destruction, we examined the p53−/−; Rag2−/−; Prkdcscid/scid triple mutant (TM) mice and p53−/−; Prkdcscid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed Rankl, encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin–Fc (rOPG-Fc) protected the bone from B-ALL–induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL–mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.

Rabbit, nutria and chinchilla testes were evaluated to compare testicular cellular senescence. There were no major species-specific differences in structure of either seminiferous tubules or interstitial tissue. There, however, were occasional abnormalities in seminiferous tubule structure with there being multinucleated and exfoliated cells present in rabbit testes. Furthermore, there were seminiferous tubules without a lumen that were filled with premeiotic/meiotic cells in nutria; and tubules with vacuolization with there being no post-meiotic cells in chinchillas. There were no differences in distribution or content of acids, total proteins and polysaccharides in the testis of any of the three species. Results using comparative immunohistochemistry procedures indicated the testes contained a few senescent cells in seminiferous tubules with typical morphology and there was a large number of senescent cells in seminiferous tubules of nutrias and chinchillas that had an abnormal structure (P

γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.

Osteoclasts are multinucleated cells responsible for bone resorption. Src homology 3 (SH3) domain-containing protein protein-2 (SH3P2)/osteoclast stimulating factor-1 regulates osteoclast differentiation, but its exact role remains elusive. Here, we show that SH3P2 suppresses osteoclast differentiation. SH3P2 knockout (KO) mice displayed decreased femoral trabecular bone mass and enhanced localization of osteoclasts on the tibial trabecular bone surface, suggesting that SH3P2 suppresses bone resorption by osteoclasts. Osteoclast differentiation based on cellular multinuclearity induced by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) was enhanced in bone marrow-derived macrophages lacking SH3P2. RANKL induced SH3P2 dephosphorylation, which increased the association of actin-dependent motor protein myosin 1E (Myo1E) with SH3P2 and thereby prevented Myo1E localization to the plasma membrane. Consistent with this, Myo1E in the membrane fraction increased in SH3P2-KO cells. Together with the attenuated osteoclast differentiation in Myo1E knocked-down cells, SH3P2 may suppress osteoclast differentiation by preventing their cell-to-cell fusion depending on Myo1E membrane localization.

Tissue engineering materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated. Traditional natural and synthetic materials are superseded by bespoke materials that cross the boundary between these two categories. Here we present hydrogels that are derived from decellularised extracellular matrix and those that are synthesised from de novo α-helical peptides. We assess in vitro activation of murine macrophages to our hydrogels and whether these gels induce an M1-like or M2-like phenotype. This was followed by the in vivo immune macrophage response to hydrogels injected into rat partial-thickness abdominal wall defects. Over 28 days we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface without promoting a foreign body reaction and see no evidence of hydrogel encapsulation or formation of multinucleate giant cells. We also note an upregulation of myogenic differentiation markers and the expression of anti-inflammatory markers Arginase1, IL-10, and CD206, indicating pro-remodelling for all injected hydrogels. Furthermore, all hydrogels promote an anti-inflammatory environment after an initial spike in the pro-inflammatory phenotype. No difference between the injected site and the healthy tissue is observed after 28 days, indicating full integration. These materials offer great potential for future applications in regenerative medicine and towards unmet clinical needs. STATEMENT OF SIGNIFICANCE: Materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated in tissue engineering. Here we present injectable hydrogels derived from decellularised extracellular matrix and de novo designed α-helical peptides. Over 28 days in the rat abdominal wall we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface with no foreign body reaction, no evidence of hydrogel encapsulation and no multinucleate giant cells. Our data indicate pro-remodelling and the promotion of an anti-inflammatory environment for all injected hydrogels with evidence of full integration with healthy tissue after 28 days. These unique materials offer great potential for future applications in regenerative medicine and towards designing materials for unmet clinical needs.

Fusion and apoptosis share a breakdown of the membrane phospholipids asymmetry, modes of which are largely unknown in osteoclastogenesis. Here, we investigated the externalization of phosphatidylserine (PS) and its receptors, and their biological functions in osteoclastogenesis. Strong immunoreactivities in vivo for the PS receptors TIM4, BAI1, and STAB2 were observed in the TRAP-positive multinucleated cells in the alveolar bone that was being remodeled around the developing dental follicles in rats. These receptors were significantly upregulated during M-CSF/RANKL-induced in vitro osteoclastogenesis using mouse bone marrow-derived cells. PS externalization in preosteoclasts was increased by the M-CSF/RANKL treatment. Multinucleation of preosteoclasts was markedly inhibited by antibodies against PS and its receptors. Among the investigated lipid transporter proteins, floppases (Abcb4, Abcc5, and Abcg1) were upregulated, whereas flippases (Atp11c and Atp8a1) downregulated during osteoclastogenesis. Preosteoclast fusion was markedly blocked by the ATPase inhibitor Na3VO4 and siRNAs against Abcc5 and Abcg1, revealing the importance of these lipid transporters in PS externalization. Further, the levels of Cd47 and Cd31, don’t-eat-me signal inducers, were increased or sustained in the early phase of osteoclastogenesis, whereas those of AnnexinI and Mfg-e8, eat-me signals inducers, were increased in the late apoptotic phase. In addition, Z-VAD-FMK, a pan caspase inhibitor, had no effect on preosteoclast fusion in the early phase of osteoclastogenesis, whereas Abs against PS, TIM4, and BAI1 decreased osteoclast apoptosis during the late phase. These results suggest that PS externalization is essential for the whole process of osteoclastogenesis and share PS receptors and transporters in the early stage fusion and late stage apoptosis. Therefore, modulation of PS and its receptors could be a useful strategy to develop anti-bone resorptive agents.

In spite of significant advancements of therapies for initial eradication of cancers, tumor relapse remains a major challenge. It is for a long time known that polyploid malignant cells are a main source of resistance against chemotherapy and irradiation. However, therapeutic approaches targeting these cells have not been appropriately pursued which could partly be due to the shortage of knowledge on the molecular biology of cell polyploidy. On the other hand, there is a rising trend to appreciate polyploid/ multinucleated cells as key players in tissue regeneration. In this review, we suggest an analogy between the functions of polyploid cells in normal and malignant tissues and discuss the idea that cell polyploidy is an evolutionary conserved source of tissue regeneration also exploited by cancers as a survival factor. In addition, polyploid cells are highlighted as a promising therapeutic target to overcome drug resistance and relapse.