Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

IL-1ß promotes osteoclastogenesis by increasing the expression of IGF2 and chemokines in non-osteoclastic cells.

Bone remodeling mediated by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCs) maintains bone structure and function. Excessive OC activation leads to bone-destroying diseases such as osteoporosis and bone erosion of rheumatoid arthritis (RA). Differentiation of OCs from bone marrow cells (BMCs) is regulated by the bone microenvironment. The proinflammatory cytokine interleukin (IL)-1ß reportedly enhances osteoclastogenesis and plays important roles in RA-associated bone loss. The present study investigated the effect of IL-1ß on OC formation via microenvironmental cells. Treating mouse BMCs with IL-1ß in the presence of receptor activator of NF-κB ligand and macrophage colony-stimulating factor increased the number of OCs. Real-time RT-PCR revealed increased expression of the IL-1ß, IL-1RI, and IL-1RII genes in non-OCs compared with OCs. Removing CD45- cells which cannot differentiate into OCs, from mouse BMCs reduced the IL-1ß-mediated enhancement of osteoclastogenesis. IL-1ß treatment upregulated the expression of inducible nitric oxide synthase, insulin-like growth factor 2 (IGF2), and the chemokines stromal cell derived factor 1, C-X3-C motif ligand 1 (CX3CL1), and CXCL7 in non-OCs. Neutralizing antibodies against these chemokines and IGF2 suppressed osteoclastogenesis in the presence of IL-1ß. These results suggest that IL-1ß enhances osteoclastogenesis by upregulating IGF2 and chemokine expression in non-OCs.

Visualization of osteocalcin and bone morphogenetic protein 2 (BMP2) secretion from osteoblastic cells by bioluminescence imaging.

The secretions of osteocalcin and bone morphogenetic protein 2 (BMP2) from living osteoblastic cells were visualized for the first time using a method of video-rate bioluminescence imaging. The fusion proteins with Gaussia luciferase (GLase) for mouse osteocalcin and BMP2 (OC-GLase and BMP2-GLase, respectively) expressed in osteoblastic MC3T3-E1 cells were correctly processed and secreted. In the video images of exocytotic secretion, the luminescence spots of OC-GLase and BMP2-GLase disappeared rapidly and gradually, respectively, indicating different manners of these proteins in diffusion. Notably, a deletion mutant of BMP2 (Δ3BMP2-GLase) lacking three basic amino acid residues in the N-terminal region for binding to heparan sulfate showed rapidly disappearing luminescence spots. In our imaging conditions, the half-life of luminescence for the spots of Δ3BMP2-GLase (1.61 ± 0.20 s) was similar to that of OC-GLase (1.22 ± 0.14 s) but not to that of BMP2-GLase (4.31 ± 0.41 s). These results suggest that, in contrast to osteocalcin, the diffusion of BMP2 from cells occurred slowly after exocytosis. Thus, our bioluminescence imaging method is useful to study the diffusion properties of secreted proteins in exocytosis.

The Inducible Nitric Oxide Synthase Pathway Promotes Osteoclastogenesis under Hypoxic Culture Conditions.

Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis.

Impacts of Glucose-Dependent Insulinotropic Polypeptide on Orthodontic Tooth Movement-Induced Bone Remodeling.

Glucose-dependent insulinotropic polypeptide (GIP) exerts extra-pancreatic effects via the GIP receptor (GIPR). Herein, we investigated the effects of GIP on force-induced bone remodeling by orthodontic tooth movement using a closed-coil spring in GIPR-lacking mice (GIPRKO) and wild-type mice (WT). Orthodontic tooth movements were performed by attaching a 10-gf nickel titanium closed-coil spring between the maxillary incisors and the left first molar. Two weeks after orthodontic tooth movement, the distance of tooth movement by coil load was significantly increased in GIPRKO by 2.0-fold compared with that in the WT. The alveolar bone in the inter-root septum from the root bifurcation to the apex of M1 decreased in both the GIPRKO and WT following orthodontic tooth movement, which was significantly lower in the GIPRKO than in the WT. The GIPRKO exhibited a significantly decreased number of trabeculae and increased trabecular separation by orthodontic tooth movement compared with the corresponding changes in the WT. Histological analyses revealed a decreased number of steady-state osteoblasts in the GIPRKO. The orthodontic tooth movement induced bone remodeling, which was demonstrated by an increase in osteoblasts and osteoclasts around the forced tooth in the WT. The GIPRKO exhibited no increase in the number of osteoblasts; however, the number of osteoclasts on the coil-loaded side was significantly increased in the GIPRKO compared with in the WT. In conclusion, our results demonstrate the impacts of GIP on the dynamics of bone remodeling. We revealed that GIP exhibits the formation of osteoblasts and the suppression of osteoclasts in force-induced bone remodeling.

Attenuation of Bone Formation through a Decrease in Osteoblasts in Mutant Mice Lacking the GM2/GD2 Synthase Gene.

The ganglioside GD1a has been reported to promote the differentiation of mesenchymal stem cells to osteoblasts in cell culture systems. However, the involvement of gangliosides, including GD1a, in bone formation in vivo remains unknown; therefore, we herein investigated their roles in GM2/GD2 synthase-knockout (GM2/GD2S KO) mice without GD1a. The femoral cancellous bone mass was analyzed using three-dimensional micro-computed tomography. A histomorphometric analysis of bone using hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase was performed to examine bone formation and resorption, respectively. Calcein double labeling was also conducted to evaluate bone formation. Although no significant differences were observed in bone mass or resorption between GM2/GD2S KO mice and wild-type (WT) mice, analyses of the parameters of bone formation using HE staining and calcein double labeling revealed less bone formation in GM2/GD2S KO mice than in WT mice. These results suggest that gangliosides play roles in bone formation.

Orthodontic tooth movement-activated sensory neurons contribute to enhancing osteoclast activity and tooth movement through sympathetic nervous signalling.

OBJECTIVES: Orthodontic tooth movement (OTM) increases sympathetic and sensory neurological markers in periodontal tissue. However, the relationship between the sympathetic and sensory nervous systems during OTM remains unclear. Therefore, the present study investigated the relationship between the sympathetic and sensory nervous systems activated by OTM using pharmacological methods. MATERIALS AND METHODS: We compared the effects of sympathectomy and sensory nerve injury during OTM in C57BL6/J mice. Capsaicin (CAP) was used to induce sensory nerve injury. Sympathectomy was performed using 6-hydroxydopamine. To investigate the effects of a ß-agonist on sensory nerve injury, isoproterenol (ISO) was administered to CAP-treated mice. Furthermore, to examine the role of the central nervous system in OTM, the ventromedial hypothalamic nucleus (VMH) was ablated using gold thioglucose. RESULTS: Sensory nerve injury and sympathectomy both suppressed OTM and decreased the percent of the alveolar socket covered with osteoclasts (Oc.S/AS) in periodontal tissue. Sensory nerve injury inhibited increases in OTM-induced calcitonin gene-related peptide (CGRP) immunoreactivity (IR), a marker of sensory neurons, and tyrosine hydroxylase (TH) IR, a marker of sympathetic neurons, in periodontal tissue. Although sympathectomy did not decrease the number of CGRP-IR neurons in periodontal tissue, OTM-induced increases in the number of TH-IR neurons were suppressed. The ISO treatment restored sensory nerve injury-inhibited tooth movement and Oc.S/AS. Furthermore, the ablation of VMH, the centre of the sympathetic nervous system, suppressed OTM-induced increases in tooth movement and Oc.S/AS. CONCLUSIONS: The present results suggest that OTM-activated sensory neurons contribute to enhancements in osteoclast activity and tooth movement through sympathetic nervous signalling.

Local administration of ReveromycinA ointment suppressed alveolar bone loss in mice.

ReveromycinA (RMA) was developed and is a unique agent for inhibiting osteoclast activity. In a previous study, we experimentally induced periodontal disease in a high-turnover osteoporosis osteoprotegerin-knockout mice (OPG KO) model and found that intraperitoneal administration of RMA inhibited alveolar bone resorption. We prepared a novel RMA-containing ointment for topical non-invasive administration in the oral cavity, in preparation for possible future clinical application. And we investigated whether this ointment can inhibit alveolar bone resorption in an experimental mouse model of periodontal disease. We examined wild-type (WT) and OPG KO mice ligated with wire around contact points on the left first and second molars to cause food impaction and induce experimental periodontal disease. RMA was administered three times a day. Using micro-computed tomography, we measured the volume of alveolar bone loss and also performed histological analysis. Our findings showed that localized administration of RMA containing ointment resulted in suppressed alveolar bone resorption, reduced osteoclast count, and lower immunostaining scores of inflammation sites compared with controls in both OPG KO and WT mice. Localized application of the specific osteoclast suppressor RMA in ointment form in the oral cavity could be a novel treatment for periodontitis that inhibits alveolar bone resorption locally.

Guanabenz inhibits alveolar bone resorption in a rat model of periodontitis.

Increase of sympathetic activity has been known to exacerbate osteoporosis through promotion of bone resorption. However, it is largely unknown about involvement of sympathetic activity in exacerbation of periodontitis. In this study, we investigated whether α2-adrenergic receptor (α2-AR) agonist guanabenz which decreases sympathetic activity, attenuates alveolar bone resorption in rats having high sympathetic activity with periodontitis. Volumes of residual alveolar bone and attachment levels in periodontium were examined using micro-computed tomography and hematoxylin-eosin staining, respectively. Furthermore, osteoclast numbers per bone surface and osteoclast surface per bone surface were measured using tartrate-resistant acid phosphatase staining. To examine the suppressive effects of guanabenz on pro-inflammatory cytokines, expression levels of tyrosine hydroxylase (TH), TNF-α, IL1-ß, and IL-6 in periodontium were measured using immunohistostaining. Administration of guanabenz attenuated loss of alveolar bone and attachment levels in rats having high sympathetic activity. Furthermore, its administration suppressed osteoclast numbers in rats having high sympathetic activity. TH, TNF-α, IL-1ß, and IL-6 positive cells in periodontium in rats treated with guanabenz for 12 weeks, were lower than those in control rats having high sympathetic activity. This study demonstrated administration of α2-AR agonist guanabenz attenuates alveolar bone resorption through decrease of sympathetic activity in rats.

Does local injection of reveromycin A inhibit tooth movement without causing systemic side effects?

OBJECTIVE: To determine the feasibility of local inhibition of osteoclast activity and control of tooth movement with local intraoral reveromycin A (RMA) injection in model mice for experimental tooth movement. MATERIALS AND METHODS: Eight-week-old wild-type mice (n = 6 per group) were divided into four groups consisting of two non-RMA groups that received normal saline for 14 (14-day non-RMA group) or 21 consecutive days (21-day non-RMA group) and 2 RMA groups that received RMA (1.0 mg/kg of weight) for 14 (14-day RMA group) or 21 consecutive days (21-day RMA group). RMA was injected locally into the buccal mucosa of the left first maxillary molar twice daily starting 3 days before placement of the 10-gf Ni-Ti closed coil spring. Tooth movement distance was analysed using micro-computed tomography. The effects on surrounding alveolar bone were evaluated by measuring the ratio of bone surface area to tissue surface area with haematoxylin-eosin-stained sections and counting the number of osteoclasts in periodontal tissue with TRAP-stained sections. Blood tests were performed and bone volume and trabecular separation at the tibial neck were measured to analyse systemic side effects. RESULTS: Local RMA injection inhibited tooth movement by 40.6 per cent, promoted alveolar bone volume maintenance by 37.4 per cent, and inhibited osteoclast activity around the tooth root at 21 days by 40.8 per cent. Systemic effects on osteoclasts or osteoblasts were not observed. CONCLUSION: Local injection of RMA enabled control of tooth movement without systemic side effects in a mouse model.

Suppression of tooth movement-induced sclerostin expression using ß-adrenergic receptor blockers.

OBJECTIVE: Regulation of bone metabolism by the sympathetic nervous system has recently been clarified. Tooth movement is increased by increased bone metabolic turnover due to sympathetic activation. This study aimed to compare the effects of the ß-adrenergic receptor (ß-AR) blockers atenolol (ß1-AR blocker), butoxamine (ß2-AR blocker) and propranolol (non-selective ß-AR blocker) on tooth movement in spontaneously hypertensive rats (SHR) with sympathicotonia. MATERIALS AND METHODS: Spontaneously hypertensive rats were divided into the following four groups: an SHR control group and groups treated with 0.1 mg/kg atenolol, 1 mg/kg butoxamine or 1 mg/kg propranolol (n = 6 rats/group). Atenolol, butoxamine or propranolol was administered daily to each treatment group, and orthodontic force was applied using a closed-coil spring. Finally, immunohistochemical analysis was performed for receptor activator of nuclear factor kappa-B ligand (RANKL) and sclerostin (SOST). RESULTS: Atenolol, butoxamine and propranolol inhibited tooth movement and increased maxillary alveolar bone volume. Histological analysis revealed that these ß-AR blockers decreased osteoclast activity on the compression side. Furthermore, immunohistochemical analysis revealed that atenolol, butoxamine and propranolol decreased the number of RANKL- and SOST-positive osteocytes on the compression side. CONCLUSIONS: ß-AR blockers decreased tooth movement and downregulated SOST in osteocytes, accompanied by increasing alveolar bone resorption.

Direct Comparison of Therapeutic Effects on Diabetic Polyneuropathy between Transplantation of Dental Pulp Stem Cells and Administration of Dental Pulp Stem Cell-Secreted Factors.

Stem cell transplantation is a potential novel therapy for diabetic polyneuropathy. Dental pulp stem cells (DPSCs) are attractive stem cell sources because DPSCs can be isolated from extracted teeth and cryopreserved while retaining viability. In this study, we directly compared the efficacy of the transplantation of DPSCs and the administration of the secreted factors from DPSCs (DPSC-SFs) on diabetic polyneuropathy. Eight weeks after streptozotocin injection, DPSCs (1.0 × 106 cells/rat) or DPSC-SFs (1.0 mL/rat) were administered into the unilateral hindlimb skeletal muscles of diabetic Sprague-Dawley rats. DPSC transplantation and DPSC-SF administration did not affect blood glucose levels and body weights in the diabetic rats. Both DPSC transplantation and DPSC-SF administration significantly ameliorated sciatic nerve conduction velocity and sciatic nerve blood flow, accompanied by increases in muscle bundle size, vascular density in the skeletal muscles and intraepidermal nerve fiber density in the diabetic rats, while there was no difference between the results for DPSCs and DPSC-SFs. These results suggest that the efficacy of both DPSC transplantation and DPSC-SF administration for diabetic polyneuropathy four weeks after transplantation/administration was mainly due to the multiple secretomes secreted from transplanted DPSCs or directly injected DPSC-SFs in the early phase of transplantation/administration.

The Effect of TBB, as an Initiator, on the Biological Compatibility of PMMA/MMA Bone Cement.

Acrylic bone cement is widely used in orthopedic surgery for treating various conditions of the bone and joints. Bone cement consists of methyl methacrylate (MMA), polymethyl methacrylate (PMMA), and benzoyl peroxide (BPO), functioning as a liquid monomer, solid phase, and polymerization initiator, respectively. However, cell and tissue toxicity caused by bone cement has been a concern. This study aimed to determine the effect of tri-n-butyl borane (TBB) as an initiator on the biocompatibility of bone cement. Rat spine bone marrow-derived osteoblasts were cultured on two commercially available PMMA-BPO bone cements and a PMMA-TBB experimental material. After a 24-h incubation, more cells survived on PMMA-TBB than on PMMA-BPO. Cytomorphometry showed that the area of cell spread was greater on PMMA-TBB than on PMMA-BPO. Analysis of alkaline phosphatase activity, gene expression, and matrix mineralization showed that the osteoblastic differentiation was substantially advanced on the PMMA-TBB. Electron spin resonance (ESR) spectroscopy revealed that polymerization radical production within the PMMA-TBB was 1/15-1/20 of that within the PMMA-BPO. Thus, the use of TBB as an initiator, improved the biocompatibility and physicochemical properties of the PMMA-based material.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3-10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2-15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

Alteration of tooth movement by reveromycin A in osteoprotegerin-deficient mice.

INTRODUCTION: Osteoprotegerin-deficient mice develop severe high-turnover osteoporosis with porous low-density trabecular bone from an age-related increase in osteoclast activity and are useful alveolar bone models of osteoporosis or frail periodontal tissue. Bisphosphonate (BP), a first-line drug for osteoporosis, is bone-avid, causing side effects such as brittle and fragile bones and jaw osteonecrosis after tooth extraction. In orthodontics, active movement is precisely controlled by temporarily suppressing and resuming movement. BP impedes such control because of its long half-life of several years in bone. Therefore, we investigated the novel osteoclast-specific inhibitor reveromycin A (RMA), which has a short half-life in bone. We hypothesized that tooth movement could be precisely controlled through temporary discontinuation and re-administration of RMA. METHODS: Osteoprotegerin-deficient mice and wild-type mice were developed as tooth movement models under constant orthodontic force. A constant orthodontic force of 10 g was induced using a nickel-titanium closed coil spring to move the maxillary first molar for 14 days. We administered BP (1.25 mg/kg) or RMA (1.0 mg/kg) continuously and then discontinued it to reveal how the subsequent movement of teeth and surrounding alveolar bone was affected. RESULTS: Continuous BP or RMA administration suppressed osteoclast activity and preserved alveolar bone around the roots, apparently normalizing bone metabolism. Tooth movement remained suppressed after BP discontinuation but resumed at a higher rate after discontinuation of RMA. CONCLUSIONS: RMA appears useful for controlling orthodontic tooth movement because it can be suppressed and resumed through administration and discontinuation, respectively.

Novel Antifungal Compound Z-705 Specifically Inhibits Protein Kinase C of Filamentous Fungi.

The cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungus Aspergillus nidulans and in MgPkc1p in the rice blast fungus Magnaporthe grisea are lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried out in silico screening by three-dimensional (3D) structural modeling and performed growth inhibition tests for M. grisea on agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed in Saccharomyces cerevisiae cells in which the kinase domain of native S. cerevisiae PKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis of MLP1, which encodes a downstream factor of PKC in S. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress in M. grisea We discuss the relationships between PKC and melanin biosynthesis.IMPORTANCE A candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified by in silico screening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed in Saccharomyces cerevisiae Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison with S. cerevisiae PKC. Analysis of the AGS1 mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungus Magnaporthe grisea revealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization in M. grisea, and this melanization, which is required for pathogenicity of M. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of 4HNR, 3HNR, and SCD1, which are essential for melanization in M. grisea, were suppressed by both PKC inhibitors.

PIEZO1 and TRPV4, which Are Distinct Mechano-Sensors in the Osteoblastic MC3T3-E1 Cells, Modify Cell-Proliferation.

Mechanical-loading and unloading can modify osteoblast functioning. Ca2+ signaling is one of the earliest events in osteoblasts to induce a mechanical stimulus, thereby demonstrating the importance of the underlying mechanical sensors for the sensation. Here, we examined the mechano-sensitive channels PIEZO1 and TRPV4 were involved in the process of mechano-sensation in the osteoblastic MC3T3-E1 cells. The analysis of mRNA expression revealed a high expression of Piezo1 and Trpv4 in these cells. We also found that a PIEZO1 agonist, Yoda1, induced Ca2+ response and activated cationic currents in these cells. Ca2+ response was elicited when mechanical stimulation (MS), with shear stress, was induced by fluid flow in the MC3T3-E1 cells. Gene knockdown of Piezo1 in the MC3T3-E1 cells, by transfection with siPiezo1, inhibited the Yoda1-induced response, but failed to inhibit the MS-induced response. When MC3T3-E1 cells were transfected with siTrpv4, the MS-induced response was abolished and Yoda1 response was attenuated. Moreover, the MS-induced response was inhibited by a TRPV4 antagonist HC-067047 (HC). Yoda1 response was also inhibited by HC in MC3T3-E1 cells and HEK cells, expressing both PIEZO1 and TRPV4. Meanwhile, the activation of PIEZO1 and TRPV4 reduced the proliferation of MC3T3-E1, which was reversed by knockdown of PIEZO1, and TRPV4, respectively. In conclusion, TRPV4 and PIEZO1 are distinct mechano-sensors in the MC3T3-E1 cells. However, PIEZO1 and TRPV4 modify the proliferation of these cells, implying that PIEZO1 and TRPV4 may be functional in the osteoblastic mechano-transduction. Notably, it is also found that Yoda1 can induce TRPV4-dependent Ca2+ response, when both PIEZO1 and TRPV4 are highly expressed.

Deficiency of GD3 Synthase in Mice Resulting in the Attenuation of Bone Loss with Aging.

Gangliosides are widely expressed in almost all tissues and cells and are also considered to be essential in the development and maintenance of various organs and tissues. However, little is known about their roles in bone metabolism. In this study, we investigated the effects of genetic deletion of ganglioside D3 (GD3) synthase, which is responsible for the generation of all b-series gangliosides, on bone metabolism. Although b-series gangliosides were not expressed in osteoblasts, these gangliosides were expressed in pre-osteoclasts. However, the expression of these gangliosides was decreased after induction of osteoclastogenesis by receptor activator of nuclear factor kappa-B ligand (RANKL). Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in GD3 synthase-knockout (GD3S KO) mice was higher than that in wild type (WT) mice at the age of 40 weeks, although there were no differences in that between GD3S KO and WT mice at 15 weeks old. Whereas bone formation parameters (osteoblast numbers/bone surface and osteoblast surface/bone surface) in GD3S KO mice did not differ from WT mice, bone resorption parameters (osteoclast numbers/bone surface and osteoclast surface/bone surface) in GD3S KO mice became significantly lower than those in WT mice at 40 weeks of age. Collectively, this study demonstrates that deletion of GD3 synthase attenuates bone loss that emerges with aging.

Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts.

Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.

Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection.

Many glycosylphosphatidylinositol-anchored proteins (GPI-APs) of fungi are membrane enzymes, organization components, and extracellular matrix adhesins. We analyzed eight Aspergillus flavus transcriptome sets for the GPI-AP gene family and identified AFLA_040110, AFLA_063860, and AFLA_113120 to be among the top 5 highly expressed genes of the 36 family genes analyzed. Disruption of the former two genes did not drastically affect A. flavus growth and development. In contrast, disruption of AFLA_113120, an orthologue of Saccharomyces cerevisiae ECM33, caused a significant decrease in vegetative growth and conidiation, promoted sclerotial production, and altered conidial pigmentation. The A. flavus ecm33 null mutant, compared with the wild type and the complemented strain, produced predominantly aflatoxin B2 but accumulated comparable amounts of cyclopiazonic acid. It showed decreased sensitivity to Congo red at low concentrations (25-50 µg/mL) but had increased sensitivity to calcofluor white at high concentrations (250-500 µg/mL). Analyses of cell wall carbohydrates indicated that the α-glucan content was decreased significantly (p < 0.05), but the contents of chitin and ß-glucan were increased in the mutant strain. In a maize colonization study, the mutant was shown to be impaired in its infectivity and produced 3- to 4-fold lower amounts of conidia than the wild type and the complemented strain. A. flavus Ecm33 is required for proper cell wall composition and plays an important role in normal fungal growth and development, aflatoxin biosynthesis, and seed colonization.