Exploring olfactory receptor family 7 subfamily C member 1 as a novel oral cancer stem cell target for immunotherapy.

The mortality rate of oral cancer has not improved over the past three decades despite remarkable advances in cancer therapies. Oral cancers contain a subpopulation of cancer stem cells (CSCs) that share characteristics associated with normal stem cells, including self-renewal and multi-differentiation potential. CSCs are tumorigenic, play a critical role in cancer infiltration, recurrence, and distant metastasis, and significantly contribute to drug resistance to current therapeutic strategies, including immunotherapy. Cytotoxic CD8+ T lymphocytes (CTLs) are key immune cells that effectively recognize peptide antigens presented by the major histocompatibility complex class I molecules. Increasing evidence suggests that cancer antigen-specific targeting by CTLs effectively regulates CSCs that drive cancer progression. In this study, we utilized data from public domains and performed various bioassays on human oral squamous cell carcinoma clinical samples and cell lines, including HSC-2 and HSC-3, to investigate the potential role of olfactory receptor family 7 subfamily C member 1 (OR7C1), a seven transmembrane G-protein-coupled olfactory receptor that is also expressed in nonolfactory tissues and was previously reported as a novel marker and target of colon cancer initiating cell-targeted immunotherapy, in CSC-targeted treatment against oral cancer. We found that the OR7C1 gene was expressed only in oral CSCs, and that CTLs reacted with human leukocyte antigen-A24-restricted OR7C1 oral CSC-specific peptides. Taken together, our findings suggest that OR7C1 represents a novel target for potent CSC-targeted immunotherapy in oral cancer.

Inhibitory effect of 405-nm blue LED light on the growth of Candida albicans and Streptococcus mutans dual-species biofilms on denture base resin.

We investigated whether irradiation with 405-nm blue LED light could inhibit the growth of not only single- but dual-species biofilms formed by Candida albicans and Streptococcus mutans on denture base resin and cause the alteration in gene expression related to adhesion and biofilm formation. C. albicans and S. mutans single-/dual-species biofilms were formed on the denture base specimens. The biofilms were irradiated with 405-nm blue LED light (power density output: 280 mW/cm2) for 0 (control) and 40 min. Dual-species biofilms were analyzed using CFU assay and fluorescence microscopy, and single-/dual-species biofilms were analyzed using alamarBlue assays and gene expression analysis. To assess the inhibitory effect of irradiation on dual-species biofilms, specimens after irradiation were aerobically incubated for 12 h. After incubation, the inhibition of growth was assessed using CFU assays and fluorescence microscopy. Data were analyzed using the Mann-Whitney U or Student's t test (p < 0.05). Irradiation produced a significant inhibitory effect on biofilms. Fluorescence microscopy revealed that almost all C. albicans and S. mutans cells were killed by irradiation, and there was no notable difference in biofilm thickness immediately after irradiation and after irradiation and incubation for 12 h. alamarBlue assays indicated the growth of the biofilms was inhibited for 12-13 h. The expression of genes associated with adhesion and biofilm formation-als1 in C. albicans and ftf, gtfC, and gtfB in S. mutans-significantly reduced by irradiation. Irradiation with 405-nm blue LED light effectively inhibited the growth of C. albicans and S. mutans dual-species biofilms for 12 h.

Beta Adrenergic Receptor Stimulation Suppresses Cell Migration in Association with Cell Cycle Transition in Osteoblasts-Live Imaging Analyses Based on FUCCI System.

Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed-ridden conditions in patients suffering from aging-associated diseases including cardiovascular, neurological, and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed-ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis are still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppress bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, fluorescent ubiquitination-based cell cycle indicator (FUCCI), in osteoblast biology and examined isoproterenol regulation of cell cycle transition and cell migration in osteoblasts. Isoproterenol treatment suppresses the levels of first entry peak of quiescent osteoblastic cells into cell cycle phase by shifting from G1 /G0 to S/G2 /M and also suppresses the levels of second major peak population that enters into S/G2 /M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1 /S nor in G2 /M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system.

PTH-Induced Osteoblast Proliferation Requires Upregulation of the Ubiquitin-Specific Peptidase 2 (Usp2) Expression.

Osteoporosis is a common disease that increases individual's fragility fracture risk. PTH is the only therapeutic agent for severe osteoporosis that requires anabolic action of bone formation. Although a part of the PTH actions is explained by increased proliferation of osteoblastic precursor cells, the mechanisms involved in the proliferation of osteoblastic cells by PTH have not been clarified yet. Therefore, in this study, we investigated the effects of PTH on gene expression in the cultured osteoblastic MC3T3-E1 cells, and found that the ubiquitin-specific peptidase 2 (Usp2) may be one of the direct target genes of PTHR signaling. Usp2 is a deubiquitination enzyme targeting various factors including CyclinD1 in cancer cells and PTH receptor 1 in osteoblasts. We confirmed that consistent induction of Usp2 expression peaked at 1 h by PTH1-34 (teriparatide) in MC3T3-E1 cells and primary calvarial osteoblasts. Among the three known splicing variants of the Usp2, we found the isoforms 1 and 2 are predominantly expressed in osteoblasts. Live-imaging analysis of the Fucci-transgenic mouse-derived primary osteoblasts indeed demonstrated that Usp2 is required for the PTH1-34-induced osteoblast proliferation. Western blotting analysis of the CyclinD1 indicated that Usp2 knock-down influences the paradoxical changes of CyclinD1 protein levels in this condition. Our data indicate that Usp2 is required for the PTH1-34-induced proliferation of osteoblasts.

PTH regulates ß2-adrenergic receptor expression in osteoblast-like MC3T3-E1 cells.

As the aged population is soaring, prevalence of osteoporosis is increasing. However, the molecular basis underlying the regulation of bone mass is still incompletely understood. Sympathetic tone acts via beta2 adrenergic receptors in bone and regulates the mass of bone which is the target organ of parathyroid hormone (PTH). However, whether beta2 adrenergic receptor is regulated by PTH in bone cells is not known. We therefore investigated the effects of PTH on beta2 adrenergic receptor gene expression in osteoblast-like MC3T3-E1 cells. PTH treatment immediately suppressed the expression levels of beta2 adrenergic receptor mRNA. This PTH effect was dose-dependent starting as low as 1 nM. PTH action on beta2 adrenergic receptor gene expression was inhibited by a transcriptional inhibitor, DRB, but not by a protein synthesis inhibitor, cycloheximide suggesting direct transcription control. Knockdown of beta2 adrenergic receptor promoted PTH-induced expression of c-fos, an immediate early response gene. With respect to molecular basis for this phenomenon, knockdown of beta2 adrenergic receptor enhanced PTH-induced transcriptional activity of cyclic AMP response element-luciferase construct in osteoblasts. Knockdown of beta2 adrenergic receptors also enhanced forskolin-induced luciferase expression, revealing that adenylate cyclase activity is influenced by beta2 adrenergic receptor. As for phosphorylation of transcription factor, knockdown of beta2 adrenergic receptor enhanced PTH-induced phosphorylation of cyclic AMP response element binding protein (CREB). These data reveal that beta2 adrenergic receptor is one of the targets of PTH and acts as a suppressor of PTH action in osteoblasts.

ß2 adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

ß adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of ß2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

Migration linked to FUCCI-indicated cell cycle is controlled by PTH and mechanical stress.

Bone metabolism is maintained via balanced repetition of bone resorption by osteoclasts and bone formation by osteoblasts. Osteoblastic cells are capable of conducting self-renewal and differentiation that are basically associated with cell-cycle transition to enable cell specification and bone formation. Osteoblasts are also migrating to fill the resorption cavity curved by osteoclasts during bone remodeling to maintain homeostasis of bone mass whose imbalance leads to osteoporosis. However, technical difficulties have hampered the research on the dynamic relationship between cell cycle and migration in osteoblasts. In this report, we overcome these problems by introducing fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter system in calvarial osteoblastic cells and reveal that the cells in G1 as well as S/G2 /M phase are migrating. Furthermore, the osteoblastic cells in S/G2 /M phase migrate faster than those in G1 phase. Interestingly, parathyroid hormone (PTH) as an anabolic agent enhances migration velocity of the cells. Mechanical stress, another anabolic signal, also enhances migration velocity. In contrast, in the presence of both PTH and mechanical stress, the migration velocity returns to the base line levels revealing the interaction between the two anabolic stimuli in the regulation of cell migration. Importantly, PTH and mechanical stress also interact when they regulate the transition of cell cycle. These data demonstrate that osteoblastic migration is linked to cell cycle and it is under the control of mechanical and chemical stimuli that coordinate to regulate bone mass.

Correlation during the extent of surgical resection, oral function and quality of life after tongue cancer surgery: Single-institution study.

INTRODUCTION: The extent of surgical resection for tongue tumors is determined by tumor size, potentially affecting oral function and quality of life (QoL). However, the relationship between oral dysfunction and QoL decline due to glossectomy extent remains unexplored. Therefore, these correlations and their predictive value for postoperative QoL decline were elucidated. METHODS: Patients treated for tongue cancer at our hospital between 2018 and 2022 were categorized by partial, hemi, or subtotal/total glossectomy. Assessments included swallowing function (RSST), articulation (Oral Diadochokinesis (ODK)), mastication, tongue pressure, and oral moisture. QoL was measured using the Oral Health Impact Profile-14 (OHIP-14). Differences within parameters were assessed using Kruskal-Wallis tests, and between-group comparisons via Mann-Whitney U tests. Spearman's correlation analysis examined parameter relationship. RESULTS: 35 patients were evaluated. Significant differences were found in ODK [ta] (p = 0.015), [ka] (p = 0.0006), tongue pressure (p = 0.0001), moisture levels (p = 0.031), OHIP-14 domains: physical disability (p = 0.014) and social disability (p = 0.046). ODK [ta] (PG: 5.95, HG: 5.38, TG: 4.03 times), [ka] (PG: 5.56, HG: 4.78, TG: 3.23 times), and tongue pressure (PG: 32.9, HG: 21.2, TG: 10.3 mmHg) decreased with glossectomy extent, while physical (PG: 0.27, HG: 2.38, TG: 2.00) and social disability (PG: 0.18, HG: 0.94, TG: 1.43) worsened. A significant negative correlation was observed between tongue pressure and social disability (p = 0.013, r = -0.36). CONCLUSION: Expanding resection significantly impacted postoperative oral function and QoL. Tongue pressure assessment may predict long-term social disability in patient QoL.

Exploring SSEA3 as an emerging biomarker for assessing the regenerative potential of dental pulp-derived stem cells.

Background: Human dental pulp-derived stem cells (hDPSCs) have emerged as a promising source for adult stem cell-based regenerative medicine. Stage-specific embryonic antigen 3 (SSEA3) is a cell surface marker associated with Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of human bone marrow-derived stem cells (hBMSCs), known for their potent regenerative potential and safety profile. In this study, we investigated the influence of the prolonged culture period and the number of culture passages on the regenerative capacity of hDPSCs and explored the association between SSEA3 expression and their regenerative abilities. Methods: hDPSCs were isolated and cultured for up to 20 passages. Cell proliferation, migration, and osteogenic, adipogenic and neurogenic differentiation potential were assessed at passages 5, 10, and 20. Flow cytometry and immunofluorescence were employed to analyze SSEA3 expression. RNA sequencing (RNA-seq) was performed on SSEA3-positive and SSEA3-negative hDPSCs to identify differentially expressed genes and associated pathways. Results: Our findings demonstrated a progressive decline in hDPSCs proliferation and migration capacity with increasing passage number. Conversely, cell size exhibited a positive correlation with passage number. Early passage hDPSCs displayed superior osteogenic and adipogenic differentiation potential. Notably, SSEA3 expression exhibited a significant negative correlation with passage numbers, reflecting the observed decline in differentiation capacity. RNA-seq analysis revealed distinct transcriptional profiles between SSEA3-positive and SSEA3-negative hDPSCs. SSEA3-positive cells displayed upregulation of genes associated with ectodermal differentiation and downregulation of genes involved in cell adhesion. Conclusions: This study elucidates the impact of passaging on hDPSC behavior and suggests SSEA3 as a valuable biomarker for evaluating stemness and regenerative potential. SSEA3-positive hDPSCs, functionally analogous to Muse cells, represent a promising cell population for developing targeted regenerative therapies with potentially improved clinical outcomes.

Metastatic gallbladder cancer presenting as numb chin syndrome: A case report and literature review.

Gallbladder cancer (GBC) is an uncommon malignancy that is highly aggressive in the advanced stages. However, it rarely metastasizes to the mandible. Numb chin syndrome (NCS) is a rare neurological manifestation associated with various underlying causes, including occult primary cancers and distant metastases. It is often considered to be a significant indicator of malignancy, and thorough investigation is essential in the presence of unclear etiology. The current study reported on the case of a 69-year-old Japanese woman who presented with numbness and mild pain in the lower lip and chin area for three months. No other systemic symptoms were observed. Immunocytochemical examination revealed the presence of an adenocarcinoma and TNM staging as per the Union for International Cancer Control and the American Joint Committee on Cancer guidelines confirmed stage IVb GBC. Comprehensive full-body positron emission tomography-computed tomography examination using 18F-fluoro-2-deoxy-D-glucose revealed additional bone and soft-tissue metastases. Palliative chemotherapy and radiation treatment were initiated based on the advanced stage of disease at the time of diagnosis. However, the patient succumbed to multiple organ failure six months later. The simultaneous occurrence of GBC, mandibular metastasis and NCS is rare and associated with poor prognosis. Despite the widespread nature of the disease, it can often manifest as non-specific oral symptoms without any systemic indications. The current study emphasizes the critical importance of timely confirmatory testing for accurate diagnosis and initiation of appropriate management for such complex conditions.

Long non-coding RNA Malat1 fine-tunes bone homeostasis and repair by orchestrating cellular crosstalk and the ß-catenin-OPG/Jagged1 pathway.

The IncRNA Malat1 was initially believed to be dispensable for physiology due to the lack of observable phenotypes in Malat1 knockout (KO) mice. However, our study challenges this conclusion. We found that both Malat1 KO and conditional KO mice in the osteoblast lineage exhibit significant osteoporosis. Mechanistically, Malat1 acts as an intrinsic regulator in osteoblasts to promote osteogenesis. Interestingly, Malat1 does not directly affect osteoclastogenesis but inhibits osteoclastogenesis in a non-autonomous manner in vivo via integrating crosstalk between multiple cell types, including osteoblasts, osteoclasts and chondrocytes. Our findings substantiate the existence of a novel remodeling network in which Malat1 serves as a central regulator by binding to ß-catenin and functioning through the ß-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In pathological conditions, Malat1 significantly promotes bone regeneration in fracture healing. Bone homeostasis and regeneration are crucial to well-being. Our discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling and repair.

Odontogenic keratocyst in the mandibular condyle base region: A case report.

Odontogenic keratocysts (OKCs) often occur in the molars in the mandibular ramus; they often progress asymptomatically and are discovered only after widespread development. Some cases of OKC progress to the mandibular condyle; however, very few cases exist only in the condyle. To the best of our knowledge, in all of the previously reported cases, OKCs occurred in the mandibular ramus, which underwent resection. The present study reports the case of a 31-year-old man in whom an OKC (13x12x6 mm) occurred discretely in the base of the condyle, in which the condylar head was successfully preserved. The tumor was removed under general anesthesia using the approach of shaving the anterior surface of the mandible. The extraction cavity was managed using the packed open technique and with an obturator. Approximately 20 months post-operation, the patient remained recurrence-free. This report presents a rare case of an OKC in the mandibular condyle base region. Resection was performed under general anesthesia and the condylar process was successfully preserved.

Exploring stage­specific embryonic antigen 3 involvement in oral cancer progression and as a potential target for taxane­based chemotherapy.

Despite significant advancements in therapeutic approaches, oral neoplasms remain formidable and life­threatening conditions that affect a substantial number of individuals worldwide. Within oral malignancies, a subset of cancer stem cells (CSCs) represent a crucial population responsible for tumor initiation and progression. The identification of reliable markers for the detection and characterization of CSCs in solid tumors, particularly in the context of oral cancers, remains an ongoing challenge. Stage­specific embryonic antigen 3 (SSEA3), previously associated with mesenchymal stem cells and linked to the progression of breast neoplasms and poor prognosis, has yet to be comprehensively elucidated in the context of oral malignancies. The present study aimed to investigate the expression and properties of SSEA3 in 16 distinct subsets of human oral neoplastic cell lines, classified as either CD44 positive (+) or CD44 negative (­). For the first time, SSEA3 was examined as an indicator of tumorigenicity and resistance to taxane­derived chemotherapeutic agents. In the majority of oral neoplastic cell lines analyzed, SSEA3 was expressed in a small population of CD44(+) cells. Significantly, SSEA3(+) cells exhibited heightened proliferative activity and upregulated expression of genes associated with stem cells compared with SSEA3(­) cells. The aforementioned findings suggested that SSEA3 may contribute to the evolution and progression of oral malignancies by fostering tumor growth. Furthermore, SSEA3(+) cells displayed increased sensitivity to taxane­based pharmaceuticals, indicating the potential for SSEA3 to be a viable target in the treatment schema for oral cavity neoplasms. In conclusion, the present study provides novel insight into the role of SSEA3 in the progression and management of oral neoplasms, potentially paving the way for more effective therapeutic approaches.

Osteolytic Bone Loss and Skeletal Deformities in a Mouse Model for Early-Onset Paget's Disease of Bone with PFN1 Mutation Are Treatable by Alendronate.

A novel osteolytic disorder due to PFN1 mutation was discovered recently as early-onset Paget's disease of bone (PDB). Bone loss and pain in adult PDB patients have been treated using bisphosphonates. However, therapeutic strategies for this specific disorder have not been established. Here, we evaluated the efficiency of alendronate (ALN) on a mutant mouse line, recapitulating this disorder. Five-week-old conditional osteoclast-specific Pfn1-deficient mice (Pfn1-cKOOCL) and control littermates (33 females and 22 males) were injected with ALN (0.1 mg/kg) or vehicle twice weekly until 8 weeks of age. After euthanizing, bone histomorphometric parameters and skeletal deformities were analyzed using 3D µCT images and histological sections. Three weeks of ALN administration significantly improved bone mass at the distal femur, L3 vertebra, and nose in Pfn1-cKOOCL mice. Histologically increased osteoclasts with expanded distribution in the distal femur were normalized in these mice. Geometric bone shape analysis revealed a partial recovery from the distal femur deformity. A therapeutic dose of ALN from 5 to 8 weeks of age significantly improved systemic bone loss in Pfn1-cKOOCL mice and femoral bone deformity. Our study suggests that preventive treatment of bony deformity in early-onset PDB is feasible.

An unusual case of oral surgical management in a patient with isovaleric acidemia and schizophrenia: A case report.

Oral/dental surgical care in patients with chronic medical comorbidities, such as isovaleric acidemia (IVA), can be challenging. In addition to technical complications, different comorbidities also present a complex range of concerning factors/challenges, which can increase the incidence of morbidity and mortality associated with surgery. IVA, a congenital error of metabolism, is a rare organic acidemia with a predisposition towards acute acidosis and life-threatening metabolic decompensation during stressful conditions, such as prolonged fasting and surgery. In addition, schizophrenia, a major neurological disorder, can result in manifestation of severe dental or periodontal conditions, including pericoronitis. The condition is associated with significant risk factors of postoperative complications, such as dangerous behaviors and adverse interactions between antipsychotic drugs and anesthetic agents. A case of comorbid dental disease with two coexisting chronic and life-threatening medical conditions, one of which is rare, is an unusual encounter in oral/dental surgery that is seldomly published. Moreover, implementing a safe and effective surgical intervention in such patients requires several informed considerations. However, only a few reported experiences or guidelines exist, reporting appropriate perioperative management strategies to minimize risks. Hence, in this case report, our experience of managing one of these rare encounters of a 20-year-old man who suffered from bilaterally partially erupted third molars, associated with chronic pericoronitis and dental caries of both the maxilla wisdom teeth with coexisting IVA and schizophrenia comorbidities is described. Additionally, the presentation and anticipated complications of the comorbid disorders of the patient are briefly reviewed. In this case, the pericoronitis and dental caries were treated by surgically removing the impacted third molars and the antagonist maxilla wisdom teeth under regional anesthesia and application of antibiotics for 3 days. The patient recovered without any postoperative complications after 1 year of follow-up.

IgSF11 regulates osteoclast differentiation through association with the scaffold protein PSD-95.

Osteoclasts are multinucleated, giant cells derived from myeloid progenitors. While receptor activator of NF-κB ligand (RANKL) stimulation is the primary driver of osteoclast differentiation, additional signaling further contributes to osteoclast maturation. Here, we demonstrate that immunoglobulin superfamily member 11 (IgSF11), whose expression increases during osteoclast differentiation, regulates osteoclast differentiation through interaction with postsynaptic density protein 95 (PSD-95), a scaffold protein with multiple protein interaction domains. IgSF11 deficiency in vivo results in impaired osteoclast differentiation and bone resorption but no observed defect in bone formation. Consequently, IgSF11-deficient mice exhibit increased bone mass. Using in vitro osteoclast culture systems, we show that IgSF11 functions through homophilic interactions. Additionally, we demonstrate that impaired osteoclast differentiation in IgSF11-deficient cells is rescued by full-length IgSF11 and that the IgSF11-PSD-95 interaction requires the 75 C-terminal amino acids of IgSF11. Our findings reveal a critical role for IgSF11 during osteoclast differentiation and suggest a role for IgSF11 in a receptor- and signal transduction molecule-containing protein complex.

Flrt2 is involved in fine-tuning of osteoclast multinucleation.

Osteoclasts are multinucleated giant cells derived from myeloid progenitors. Excessive bone resorption by osteoclasts can result in serious clinical outcomes for which better treatment options are needed. Here, we identified fibronectin leucine-rich transmembrane protein 2 (Flrt2), a ligand of the Unc5 receptor family for neurons, as a novel target associated with the late/maturation stage of osteoclast differentiation. Flrt2 expression is induced by stimulation with receptor activator of nuclear factor-kB ligand (RANKL). Flrt2 deficiency in osteoclasts results in reduced hyper-multinucleation, which could be restored by RNAi-mediated knockdown of Unc5b. Treatment with Netrin1, another ligand of Unc5b which negatively controls osteoclast multinucleation through down regulation of RANKL-induced Rac1 activation, showed no inhibitory effects on Flrt2-deficient cells. In addition, RANKL-induced Rac1 activation was attenuated in Flrt2-deficient cells. Taken together, these results suggest that Flrt2 regulates osteoclast multinucleation by interfering with Netrin 1-Unc5b interaction and may be a suitable therapeutic target for diseases associated with bone remodeling. [BMB Reports 2019; 52(8): 514-519].

Profilin 1 Negatively Regulates Osteoclast Migration in Postnatal Skeletal Growth, Remodeling, and Homeostasis in Mice.

Profilin 1 (Pfn1), a regulator of actin polymerization, controls cell movement in a context-dependent manner. Pfn1 supports the locomotion of most adherent cells by assisting actin-filament elongation, as has been shown in skeletal progenitor cells in our previous study. However, because Pfn1 has also been known to inhibit migration of certain cells, including T cells, by suppressing branched-end elongation of actin filaments, we hypothesized that its roles in osteoclasts may be different from that of osteoblasts. By investigating the osteoclasts in culture, we first verified that Pfn1-knockdown (KD) enhances bone resorption in preosteoclastic RAW264.7 cells, despite having a comparable number and size of osteoclasts. Pfn1-KD in bone marrow cells showed similar results. Mechanistically, Pfn1-KD osteoclasts appeared more mobile than in controls. In vivo, the osteoclast-specific conditional Pfn1-deficient mice (Pfn1-cKO) by CathepsinK-Cre driver demonstrated postnatal skeletal phenotype, including dwarfism, craniofacial deformities, and long-bone metaphyseal osteolytic expansion, by 8 weeks of age. Metaphyseal and diaphyseal femurs were drastically expanded with suppressed trabecular bone mass as indicated by µCT analysis. Histologically, TRAP-positive osteoclasts were increased at endosteal metaphysis to diaphysis of Pfn1-cKO mice. The enhanced movement of Pfn1-cKO osteoclasts in culture was associated with a slight increase in cell size and podosome belt length, as well as an increase in bone-resorbing activity. Our study, for the first time, demonstrated that Pfn1 has critical roles in inhibiting osteoclast motility and bone resorption, thereby contributing to essential roles in postnatal skeletal homeostasis. Our study also provides novel insight into understanding skeletal deformities in human disorders.