The histone acetyltransferase Mof regulates Runx2 and Osterix for osteoblast differentiation.

Osteoblast differentiation is regulated by various transcription factors, signaling molecules, and posttranslational modifiers. The histone acetyltransferase Mof (Kat8) is involved in distinct physiological processes. However, the exact role of Mof in osteoblast differentiation and growth remains unknown. Herein, we demonstrated that Mof expression with histone H4K16 acetylation increased during osteoblast differentiation. Inhibition of Mof by siRNA knockdown or small molecule inhibitor, MG149 which is a potent histone acetyltransferase inhibitor, reduced the expression level and transactivation potential of osteogenic key markers, Runx2 and Osterix, thus inhibiting osteoblast differentiation. Besides, Mof overexpression also enhanced the protein levels of Runx2 and Osterix. Mof could directly bind the promoter region of Runx2/Osterix to potentiate their mRNA levels, possibly through Mof-mediated H4K16ac to facilitate the activation of transcriptional programs. Importantly, Mof physically interacts with Runx2/Osterix for the stimulation of osteoblast differentiation. Yet, Mof knockdown showed indistinguishable effect on cell proliferation or apoptosis in MSCs and preosteoblast cells. Taken together, our results uncover Mof functioning as a novel regulator of osteoblast differentiation via the promotional effects on Runx2/Osterix and rationalize Mof as a potential therapeutic target, like possible application of inhibitor MG149 for the treatment of osteosarcoma or developing specific Mof activator to ameliorate osteoporosis.

Eldecalcitol inhibits the progression of oral cancer by suppressing the expression of GPx-1.

OBJECTIVES: This study aimed to investigate the role of eldecalcitol in the progression of oral squamous cell carcinoma and to explore the related mechanism. MATERIALS AND METHODS: The effects of eldecalcitol on the proliferation, cell cycle, apoptosis, and migration of oral cancer cells (SCC-15 and CAL-27) were evaluated with cell counting kit-8, flow cytometry, quantitative real-time polymerase chain reaction, western blotting, and scratch assay. Mouse xenograft tumor model was established to further confirm the role of eldecalcitol in the progression of oral cancer. Immunohistochemistry, quantitative real-time polymerase chain reaction, and western blotting were used to detect glutathione peroxidase-1 expression in oral cancer tissue and cells treated with eldecalcitol. RESULTS: Eldecalcitol was found to inhibit the proliferation and migration of SCC-15 and CAL-27 cells significantly, block the cell cycle in the G0/G1 phase, and enhance the apoptosis. In addition, glutathione peroxidase-1 was downregulated by eldecalcitol and acted as an important medium of eldecalcitol in inhibiting the proliferation and migration of SCC-15 and CAL-27 cells, as well as promoting their apoptosis. CONCLUSIONS: Eldecalcitol may inhibit the progression of oral cancer by suppressing the expression of glutathione peroxidase-1, which may provide new insight into the application of eldecalcitol as a potential anti-cancer drug.

Periostin regulates LPS-induced apoptosis via Nrf2/HO-1 pathway in periodontal ligament fibroblasts.

OBJECTIVE: Periostin is important for the maintenance of periodontal tissue, but its role in periodontitis is controversial. This research investigated the effect of periostin in periodontitis and the underlying mechanism. DESIGN: Mouse periodontitis models in vivo and inflammation model in vitro which were induced by Porphyromonas gingivalis lipopolysaccharide were established to evaluate periostin expression. Human periodontal ligament fibroblasts (PDLFs) were treated with lipopolysaccharide and N-acetylcysteine, fluorescence staining, flow cytometry, Western blot, and qRT-PCR were used to detect reactive oxygen species (ROS), periostin expression, and apoptosis-related makers. The periostin gene was successfully transfected into PDLFs to verify the effect of periostin on apoptosis. Then, the Nrf2 inhibitor was added to clarify the mechanism. RESULTS: Periostin expression decreased in the periodontal ligaments of mouse periodontitis models and lipopolysaccharide-induced PDLFs. Lipopolysaccharide promoted the activation of ROS and apoptosis in PDLFs, whereas N-acetylcysteine reversed this condition. Overexpression of periostin suppressed apoptosis of PDLFs and reversed the inhibitory effect of lipopolysaccharide on nuclear Nrf2 expression. Moreover, the Nrf2 inhibitor attenuated the protective effect of periostin on lipopolysaccharide-induced apoptosis. CONCLUSIONS: Lipopolysaccharide induced apoptosis in PDLFs by inhibiting periostin expression and thus Nrf2/HO-1 pathway, indicating that periostin could be a potential therapeutic target for periodontitis.

Regulatory T cells showed characteristics of T helper-17(Th17) cells in mice periodontitis model.

OBJECTIVES: This study aimed to clarify the regulatory role of Th17-Treg balance in periodontitis and further reveal Treg plasticity. MATERIALS AND METHODS: An experimental periodontitis model was established by ligation and injection of Pg-LPS. Inflammatory factors were measured by ELISA and RT-PCR. Alveolar bone absorption was evaluated by micro-CT and histomorphology. Quantities of Treg and Th17 cell and their related gene expression were examined. Furthermore, after magnetic bead-sorting spleen Treg cells, Treg/Th17 characteristic genes were explored. Immunofluorescence double staining of Foxp3 and IL-17 was conducted to further reveal Treg plasticity. RESULTS: Inflammatory cytokines in serum and gingival tissue increased significantly in periodontitis, which revealed obvious crestal bone loss. Further analysis showed that the number of Th17 cells and expression of related genes increased more significantly than Treg cells, demonstrating Treg-Th17 imbalance. Flow cytometry showed that the proportions of Treg cells in the blood and spleen were lower in periodontitis group. Furthermore, Foxp3 was downregulated, and Rorc/ IL-17A were increased in Treg cells of periodontitis group. Immunofluorescence double staining showed significantly increased number of IL-17+Foxp3+ cells in periodontitis. CONCLUSIONS: These results provided evidence that Treg cells showed characteristics of Th17 cells in mice with periodontitis, although its mechanisms require further study.

Immunolocalization of Enzymes/Membrane Transporters Related to Bone Mineralization in the Metaphyses of the Long Bones of Parathyroid-Hormone-Administered Mice.

The present study aimed to demonstrate the immunolocalization and/or gene expressions of the enzymes and membrane transporters involved in bone mineralization after the intermittent administration of parathyroid hormone (PTH). The study especially focused on TNALP, ENPP1, and PHOSPHO1, which are involved in matrix vesicle-mediated mineralization, as well as PHEX and the SIBLING family, which regulate mineralization deep inside bone. Six-week-old male mice were subcutaneously injected with 20 µg/kg/day of human PTH (1-34) two times per day (n = 6) or four times per day (n = 6) for two weeks. Additionally, control mice (n = 6) received a vehicle. Consistently with an increase in the volume of the femoral trabeculae, the mineral appositional rate increased after PTH administration. The areas positive for PHOSPHO1, TNALP, and ENPP1 in the femoral metaphyses expanded, and the gene expressions assessed by real-time PCR were elevated in PTH-administered specimens when compared with the findings in control specimens. The immunoreactivity and/or gene expressions of PHEX and the SIBLING family (MEPE, osteopontin, and DMP1) significantly increased after PTH administration. For example, MEPE immunoreactivity was evident in some osteocytes in PTH-administered specimens but was hardly observed in control specimens. In contrast, mRNA encoding cathepsin B was significantly reduced. Therefore, the bone matrix deep inside might be further mineralized by PHEX/SIBLING family after PTH administration. In summary, it is likely that PTH accelerates mineralization to maintain a balance with elevated matrix synthesis, presumably by mediating TNALP/ENPP1 cooperation and stimulating PHEX/SIBLING family expression.

The Synergistic Effect of Zuogui Pill and Eldecalcitol on Improving Bone Mass and Osteogenesis in Type 2 Diabetic Osteoporosis.

Background and Objectives: The incidence of diabetic osteoporosis, an important complication of diabetes mellitus, is increasing gradually. This study investigated the combined effect of the Zuogui pill (ZGP) and eldecalcitol (ED-71), a novel vitamin D analog, on type 2 diabetic osteoporosis (T2DOP) and explored their action mechanism. Materials and Methods: Blood glucose levels were routinely monitored in db/db mice while inducing T2DOP. We used hematoxylin and eosin staining, Masson staining, micro-computed tomography, and serum biochemical analysis to evaluate changes in the bone mass and blood calcium and phosphate levels of mice. Immunohistochemical staining was performed to assess the osteoblast and osteoclast statuses. The MC3T3-E1 cell line was cultured in vitro under a high glucose concentration and induced to undergo osteogenic differentiation. Quantitative real-time polymerase chain reaction, Western blot, immunofluorescence, ALP, and alizarin red staining were carried out to detect osteogenic differentiation and PI3K-AKT signaling pathway activity. Results: ZGP and ED-71 led to a dramatic decrease in blood glucose levels and an increase in bone mass in the db/db mice. The effect was strongest when both were used together. ZGP combined with ED-71 promoted osteoblast activity and inhibited osteoclast activity in the trabecular bone region. The in vitro results revealed that ZGP and ED-71 synergistically promoted osteogenic differentiation and activated the PI3K-AKT signaling pathway. The PI3K inhibitor LY294002 or AKT inhibitor ARQ092 altered the synergistic action of both on osteogenic differentiation. Conclusions: The combined use of ZGP and ED-71 reduced blood glucose levels in diabetic mice and promoted osteogenic differentiation through the PI3K-AKT signaling pathway, resulting in improved bone mass. Our study suggests that the abovementioned combination constitutes an effective treatment for T2DOP.

Dapagliflozin, sildenafil and their combination in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Dapagliflozin, a selective inhibitor of sodium-glucose cotransporter 2 (SGLT2), can reduce cardiovascular events and mortality in patients with heart failure. A number of mechanisms have been proposed to explain the beneficial effects of SGLT2 inhibitors. The purpose of this study was to determine whether dapagliflozin can improve pulmonary vascular remodelling and the efficacy of dapagliflozin as an add-on therapy to sildenafil in rats with pulmonary arterial hypertension (PAH). METHODS: A monocrotaline (MCT)-induced PAH rat model was used in our study. MCT-injected rats were randomly divided into four groups and treated for 3 weeks with daily per os treatment with vehicle, dapagliflozin (1 mg/kg/day), sildenafil (25 mg/kg/day), or a combination of dapagliflozin (1 mg/kg/day) and sildenafil (25 mg/kg/day). Haemodynamic measurements, histological analysis, enzyme-linked immunosorbent assay and western blotting analysis were employed to detect the changes in PAH rats after treatments. RESULTS: Dapagliflozin significantly attenuated MCT-induced increases in right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) in PAH rats. Dapagliflozin effectively decreased the thickening of pulmonary artery media and decreased the muscularization of pulmonary arterioles in PAH rats. Moreover, dapagliflozin attenuated nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation in lung tissues and the levels of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in plasma. However, dapagliflozin as an add-on therapy to sildenafil in rats with PAH did not show a more pronounced beneficial effect on right ventricular systolic pressure and pulmonary vascular remodelling in MCT rats than sildenafil alone. CONCLUSIONS: Dapagliflozin reduces right ventricular systolic pressure and pulmonary vascular remodelling in a rat model of PAH. However, combination therapy with dapagliflozin and sildenafil was not more effective than monotherapy with sildenafil in PAH rats.

m6A regulator-mediated RNA methylation modification patterns are involved in immune microenvironment regulation of periodontitis.

The role of epigenetic regulation in immunity is emerging, especially for RNA N6-methyladenosine (m6A) modification. However, little is known about the role of m6A in the regulation of the immune microenvironment of periodontitis. Thus, we aim to investigate the impact of m6A modification in periodontitis immune microenvironment. The RNA modification patterns mediated by 23 m6A-regulators were systematically evaluated in 310 periodontitis samples. The impact of m6A modification on immune microenvironment characteristics was explored, including infiltrating immunocytes, immune reaction gene-sets and HLAs (human leukocyte antigen) gene. m6A phenotype-related immune genes were also identified. 17 m6A regulators were dysregulated and a 15-m6A regulator signature can well distinguish periodontitis and control samples. ALKBH5 and FMR1 are closely related to infiltrating monocyte abundance. ELAVL1 and CBLL1 are significant regulators in immune reaction of TNF_Family_Members_Receptors and Cytokine. The expression of HLA-B and HLA-DOA is affected by ALKBH5 and LRPPRC. 3 distinct RNA modification patterns mediated by 23 m6A regulators were identified. They differ from immunocyte abundance, immune reaction and HLA gene. 1631 m6A phenotype-related genes and 70 m6A-mediated immune genes were identified, and the biological functions of these were explored. Our finding demonstrated the m6A modification plays a crucial role in the diversity and complexity of the immune microenvironment of periodontitis.

Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone.

In this study, we examined the immunolocalization of podoplanin/E11, CD44, actin filaments, and phosphorylated ezrin in the osteoblasts on the verge of differentiating into osteocytes in murine femora and tibiae. When observing under stimulated emission depletion microscopy, unlike podoplanin-negative osteoblasts, podoplanin-positive osteoblasts showed a rearranged assembly of actin filaments along the cell membranes which resembled that of embedded osteocytes. In the metaphysis, i.e., the bone remodeling site, CD44-bearing osteoclasts were either proximal to or in contact with podoplanin-positive osteoblasts, but the podoplanin-positive osteoblasts also localized CD44 on their own cell surface. These podoplanin-positive osteoblasts, which either possessed CD44 on their cell surface or were close to CD44-bearing osteoclasts, showed phosphorylated ezrin-positivity on the cell membranes. Therefore, the CD44/podoplanin interaction on the cell surface may be involved in the osteoblastic differentiation into osteocytes in the metaphyses, via the mediation of podoplanin-driven ezrin phosphorylation and the subsequent reorganized assembly of actin filaments. Consistently, the protein expression of phosphorylated ezrin was increased after CD44 administration in calvarial culture. Conversely, in modeling sites such as the cortical bones, podoplanin-positive osteoblasts were uniformly localized at certain intervals even without contact with CD44-positive bone marrow cells; furthermore, they also exhibited phosphorylated ezrin immunoreactivity along their cell membranes. Taken together, it seems likely that the CD44/podoplanin interaction is involved in osteoblastic differentiation into osteocytes in the bone remodeling area but not in modeling sites.

Intermittent PTH Administration Increases Bone-Specific Blood Vessels and Surrounding Stromal Cells in Murine Long Bones.

To verify whether PTH acts on bone-specific blood vessels and on cells surrounding these blood vessels, 6-week-old male mice were subjected to vehicle (control group) or hPTH [1-34] (20 µg/kg/day, PTH group) injections for 2 weeks. Femoral metaphyses were used for histochemical and immunohistochemical studies. In control metaphyses, endomucin-positive blood vessels were abundant, but αSMA-reactive blood vessels were scarce. In the PTH-administered mice, the lumen of endomucin-positive blood vessels was markedly enlarged. Moreover, many αSMA-positive cells were evident near the blood vessels, and seemed to derive from those vessels. These αSMA-positive cells neighboring the blood vessels showed features of mesenchymal stromal cells, such as immunopositivity for c-kit and tissue nonspecific alkaline phosphatase (TNALP). Thus, PTH administration increased the population of perivascular/stromal cells positive for αSMA and c-kit, which were likely committed to the osteoblastic lineage. To understand the cellular events that led to increased numbers and size of bone-specific blood vessels, we performed immunohistochemical studies for PTH/PTHrP receptor and VEGF. After PTH administration, PTH/PTHrP receptor, VEGF and its receptor flk-1 were consistently identified in both osteoblasts and blood vessels (endothelial cells and surrounding perivascular cells). Our findings suggest that exogenous PTH increases the number and size of bone-specific blood vessels while fostering perivascular/stromal cells positive for αSMA/TNALP/c-kit.

Altered immunolocalization of FGF23 in murine femora metastasized with human breast carcinoma MDA-MB-231 cells.

INTRODUCTION: After the onset of bone metastasis, tumor cells appear to modify surrounding microenvironments for their benefit, and particularly, the levels of circulating fibroblast growth factor (FGF) 23 in patients with tumors have been highlighted. MATERIALS AND METHODS: We have attempted to verify if human breast carcinoma MDA-MB-231 cells metastasized in the long bone of nu/nu mice would synthesize FGF23. Serum concentrations of calcium, phosphate (Pi) and FGF23 were measured in control nu/nu mice, bone-metastasized mice, and mice with mammary gland injected with MDA-MB-231 cells mimicking primary mammary tumors. RESULTS AND CONCLUSIONS: MDA-MB-231 cells revealed intense FGF23 reactivity in metastasized lesions, whereas MDA-MB-231 cells cultured in vitro or when injected into the mammary glands (without bone metastasis) showed weak FGF23 immunoreactivity. Although the bone-metastasized MDA-MB-231 cells abundantly synthesized FGF23, osteocytes adjacent to the FGF23-immunopositive tumors, unlike intact osteocytes, showed no FGF23. Despite significantly elevated serum FGF23 levels in bone-metastasized mice, there was no significant decrease in the serum Pi concentration when compared with the intact mice and mice with a mass of MDA-MB-231 cells in mammary glands. The metastasized femora showed increased expression and FGFR1 immunoreactivity in fibroblastic stromal cells, whereas femora of control mice showed no obvious FGFR1 immunoreactivity. Taken together, it seems likely that MDA-MB-231 cells synthesize FGF23 when metastasized to a bone, and thus affect FGFR1-positive stromal cells in the metastasized tumor nest in a paracrine manner.

Fine mapping of qWCR7, a grain chalkiness QTL in rice.

Chalkiness is one of the key determinants of rice quality and is a highly undesirable trait for breeding and marketing. In this study, qWCR7, a major quantitative trait locus (QTL) of white-core rate (WCR), was genetically validated using a BC3F2 segregation population and further fine mapped using a near isogenic line (NIL) population, of which both were derived from a cross between the donor parent DL208 and the recurrent parent ZS97. qWCR7 was finally narrowed to a genomic interval of ~ 68 kb, containing seven annotated genes. Among those, two genes displayed markedly different expression levels in endosperm of NILs. Transcriptome analysis showed that the synthesis and accumulation of metabolites played a key role in chalkiness formation. The contents of storage components and expression levels of related genes were detected, suggesting that starch and storage protein were closely related to white-core trait. Our findings have laid the foundation of map-based cloning of qWCR7, which may have potential value in quality improvement during rice breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01260-x.

Immune landscape of periodontitis unveils alterations of infiltrating immunocytes and molecular networks-aggregating into an interactive web-tool for periodontitis related immune analysis and visualization.

BACKGROUND: Immunity reaction plays an essential role in periodontitis progress and we aim to investigate the underlying regulatory network of immune reactions in periodontitis. METHODS: CIBERSORT was used to estimate immunocyte fractions in different clinical statuses. Logistic regression was used to assess the immunocyte weight in periodontitis. Immune-related periodontitis subtypes were identified by the Nonnegative Matrix Factorization algorithm. Gene-set enrichment analysis and Gene-set variation analysis were conducted to analyze pathway activities. Immunocytes related gene modules were identified by Weighted gene co-expression network analysis. RESULTS: Altered immunocytes in healthy versus periodontitis, aggressive versus chronic, male versus female and age were identified. Immunocytes enriched in periodontitis were calculated, and their correlation was also explored. Two distinct immune-related periodontitis subtypes were identified and one is characterized by B cell reactions and the other is IL-6 cytokine reactions. 463 statistically significant correlations between 22 immunocytes and pathways were revealed. Immunocytes and clinical phenotypes matched their gene modules, and their functions were annotated. Last, an easy-to-use and user-friendly interactive web-tool were developed for periodontitis related immune analysis and visualization ( https://118.24.100.193:3838/tool-PIA/ ). CONCLUSIONS: This study systematically investigated periodontitis immune atlas and caught a glimpse of the underlying mechanism of periodontitis from gene-pathway-immunocyte networks, which can not only inspire researchers but also help them in periodontitis related immune researches.

Fusion and hemagglutinin proteins of canine distemper virus promote osteoclast formation through NF-κB dependent and independent mechanisms.

Paget's disease (PD) features abnormal osteoclasts (OC) which sharply increase in number and size and then intensely induce bone resorption. The purpose of this study was to determine the direct effects of canine distemper virus (CDV) and its fusion protein and hemagglutinin protein (F + H) on receptor activator of nuclear factor kappa-B ligand (RANKL) induced OC formation in vitro. Immunofluorescence assay, OC morphological and functional detection, intracellular signaling pathway detection, Real-time PCR analysis and ELISA were applied in this study. Immunofluorescence assay provided the conclusive proof that CDV can infect and replicate in RAW264.7 mouse monocyte cell line, primary human peripheral blood mononuclear cells (PBMC) and their further fused OC. Both CDV and F + H significantly promoted OC formation and bone resorption ability induced by RANKL. Meanwhile, intracellular signaling transduction analysis revealed CDV and F + H specifically upregulated the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) induced by RANKL, respectively. Furthermore, without RANKL stimulation, both CDV and F + H slightly induced OC-like cells formation in RAW264.7 cell line even in the presence of NF-κB inhibitor. F + H upregulate OC differentiation and activity through modulation of NF-κB signaling pathway, and induce OC precursor cells merging dependent on the function of glycoproteins themselves. These results meant that F and H proteins play a pivotal role in CDV supporting OC formation. Moreover, this work further provide a new research direction that F and H proteins in CDV should be considered as a trigger during the pathogenesis of PD.

Silencing FOXO1 attenuates dexamethasone-induced apoptosis in osteoblastic MC3T3-E1 cells.

Dexamethasone (DEX), a widely used glucocorticoid with strong anti-inflammatory and immunosuppressive activities, has been reported to induce apoptosis in osteoblasts, but the underlying mechanisms are still not comprehensively investigated. FOXO1 plays an important role in the regulation of cell proliferation and apoptosis. Our study aims to explore the role of FOXO1 in DEX-induced apoptosis of osteoblastic MC3T3-E1 cells through bioinformatics and experiments. We first employed bioinformatics to identify DEX-related genes and revealed their functions by GO enrichment analysis including FOXO1 associated biological processes. Expression level of FOXO1 was validated by GEO data. Then, experiments were performed to verify the hypothesis. CCK8 was used to detect cell viability and apoptosis was detected by flow cytometry. SiRNA was used to silence FOXO1 and western-blot was employed to detect protein expression. Results demonstrated DEX-related genes involved in cell proliferation, apoptosis and angiogenesis and FOXO1 was a regulator of apoptosis. DEX could up-regulate FOXO1 expression, inhibit cell viability, promote apoptosis. SiRNA-FOXO1 could attenuate DEX-induced apoptosis in MC3T3-E1. These findings suggested DEX could affect some vital biological processes of MC3T3-E1 and FOXO1 played an essential role in DEX-induced apoptosis in MC3T3-E1.

Functions of Periostin in Dental Tissues and Its Role in Periodontal Tissue Regeneration.

The goal of periodontal regeneration therapy is to reliably restore teeth's supporting periodontal tissue, while aiding the formation of new connective tissue attached to the periodontal ligament (PDL) fibers and new alveolar bone. Periostin is a matricellular protein, primarily expressed in the periosteum and PDL of adult mice. Its biological functions have been extensively studied in the fields of cardiovascular physiology and oncology. Despite being initially identified in bone and dental tissue, the function of Periostin in PDL and the pathophysiology associated with alveolar bone are scarcely studied. Recently, several studies have suggested that Periostin may be an important regulator of periodontal tissue formation. By promoting collagen fibrillogenesis and the migration of fibroblasts and osteoblasts, Periostin might play a key role in the regeneration of PDL and alveolar bone after periodontal surgery. In this chapter, the implications of Periostin in periodontal tissue biology and its potential use in periodontal tissue regeneration are reviewed.

Diallyl Disulfide Induces Apoptosis and Autophagy in Human Osteosarcoma MG-63 Cells through the PI3K/Akt/mTOR Pathway.

Diallyl disulfide (DADs), a natural organic compound, is extracted from garlic and scallion and has anti-tumor effects against various tumors. This study investigated the anti-tumor activity of DADs in human osteosarcoma cells and the mechanisms. MG-63 cells were exposed to DADs (0, 20, 40, 60, 80, and 100 µM) for different lengths of time (24, 48, and 72 h). The CCK8 assay results showed that DADs inhibited osteosarcoma cell viability in a dose-and time-dependent manner. FITC-Annexin V/propidium iodide staining and flow cytometry demonstrated that the apoptotic ratio increased and the cell cycle was arrested at the G2/M phase as the DADs concentration was increased. A Western blot analysis was employed to detect the levels of caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and p62 as well as suppression of the mTOR pathway. High expression of LC3-II protein revealed that DADs induced formation of autophagosome. Furthermore, DADs-induced apoptosis was weakened after adding 3-methyladenine, demonstrating that the DADs treatment resulted in autophagy-mediated death of MG-63 cells. In addition, DADs depressed p-mTOR kinase activity, and the inhibited PI3K/Akt/mTOR pathway increased DADs-induced apoptosis and autophagy. In conclusion, our results reveal that DADs induced G2/M arrest, apoptosis, and autophagic death of human osteosarcoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

Inflammation, mesenchymal stem cells and bone regeneration.

Achieving satisfactory reconstruction of bone remains an important goal in orthopedic and dental conditions such as bone trauma, osteoporosis, arthritis, osteonecrosis, and periodontitis. Appropriate temporal and spatial differentiation of mesenchymal stem cells (MSCs) is essential for postnatal bone regeneration. Additionally, an acute inflammatory response is crucial at the onset of bone repair, while an adaptive immune response has important implications during late bone remodeling. Various reports have indicated bidirectional interactions between MSCs and inflammatory cells or molecules. For example, inflammatory cells can recruit MSCs, direct their migration and differentiation, so as to exert anabolic effects on bone repair. Furthermore, both pro-inflammatory and anti-inflammatory cytokines can regulate MSCs properties and subsequent bone regeneration. MSCs have demonstrated highly immunosuppressive functions, such as inhibiting the differentiation of monocytes/hematopoietic precursors and suppressing the secretion of pro-inflammatory cytokines. This review emphasizes the important interactions between inflammatory stimuli, MSCs, and bone regeneration as well as the underlying regulatory mechanisms. Better understanding of these principles will provide new opportunities for promoting bone regeneration and the treatment of bone loss associated with immunological diseases.

Histochemical evidence of IGF2 mRNA-binding protein 2-mediated regulation of osteoclast function and adhesive ability.

Insulin-like growth factor 2 (IGF2) messenger RNA-binding proteins (IMPs) are a family of oncofetal RNA-binding proteins that play important roles in cell migration, renewal, and metabolism. IMP2 gene expression may be important in determining IGF2 levels and might, thereby, be central to bone metabolism. In our present study, IMP2-deficient mice exhibited more immature bone structures, characterized by abundant residual cartilage cores; growth plates containing more rich cartilage matrix, which was arranged irregularly; and a significantly thicker hypertrophic chondrocyte layer in the femoral metaphysis, compared with wild-type mice. These abnormalities were associated with profound effects on the size and morphology of osteoclasts. Specifically, the osteoclasts exhibited various polymorphisms, failed to form resorption lacunae, and were detached from the bone surface. Consistent with these findings, IMP2 deficiency reduced the expression of two important proteases (cathepsin K and matrix metallopeptidase 9) as well as that of C-SRC, a critical regulator of ruffled border formation in osteoclasts, indicating impaired osteoclastic activity. IMP2-deficient mice also displayed inhibited osteoclast adhesion owing to defects in the CD44-osteopontin signaling pathway. In summary, we used IMP2-deficient mice as a model to determine whether IMP2 plays a role during bone metabolism. Our results indicate that IMP2 deficiency delayed bone remodeling by significantly inhibiting the activity of osteoclasts and impairing their adhesion.

Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM).

The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes' cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.