Runx2 Protein Levels Research Articles

Overexpression of miR-182 inhibits ossification of ligamentum flavum cells by targeting NAMPT

Ossification of the ligamentum flavum (OLF) is a debilitating disease resulting from the development of ectopic bone formation, which leads to the compression of the spinal cord. Nicotinamide phosphoribosyltransferase (NAMPT) was found to be upregulated and microRNA-182 (miR-182) was downregulated in OLF tissue. We investigated the effects of NAMPT and miR-182 expression in OLF cells and the influence on proteins associated with osteogenic differentiation. MiR-182 overexpression inhibited NAMPT, RUNX2, OCN and OPN mRNA and protein expression in OLF cells. Alkaline phosphatase (ALP) assay and Alizarin red staining confirmed reduced levels of osteogenic differentiation and mineralized nodule formation. Knockdown of NAMPT and the NAMPT inhibitor FK866 also inhibits RUNX2, OCN and OPN mRNA expression and protein levels, whereas overexpression of NAMPT promotes the expression of RUNX2, OCN and OPN and the generation of bone nodules. Dual-luciferase reporter assays revealed that miR-182 directly targets NAMPT and downregulates its expression. Transfection of OLF cells with miR-182 downregulated NAMPT and suppressed the regulation of RUNX2, OCN, and OPN by NAMPT overexpression. Overall, these data demonstrate that miR-182 suppresses OLF by downregulating NAMPT.

Carbon monoxide releasing molecule‑3 promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells by releasing carbon monoxide.

Stem cell‑based therapies are promising strategies to stimulate bone regeneration.Carbon monoxide releasing molecule‑3 (CORM‑3) exhibits multiple regulatory effects in a number of cells by releasing carbon monoxide (CO). The present study aimed to investigate the influence of CORM‑3 on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were divided into five groups: A CORM‑3‑osteogenic group, in which cells were pretreated with CORM‑3 and subjected to osteogenic differentiation induction using osteogenic medium; an osteogenic group, in which cells were cultured in osteogenic medium; a degassed CORM‑3‑osteogenic group, in which cells were pretreated with degassed CORM‑3 and subjected to osteogenic differentiation induction; a CORM‑3 group, in which cells were cultured in control medium containing CORM‑3; and a control group, in which cells were cultured in control medium alone. The osteo‑specific mRNA and protein expression of runt‑related transcription factor2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) were assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Alkaline phosphatase (ALP) activity was also examined and mineralization was detected using alizarin red staining. Levels of Runx2, OCN and OPN mRNA and protein in the CORM‑3‑osteogenic group were significantly increased compared with the osteogenic group (P<0.05), with the exception of OCN protein levels on day3. The mRNA and protein expression of Runx2, OCN and OPN in the degassed CORM‑3‑osteogenic and osteogenic groups were similar. In addition, the mRNA and protein expression of Runx2, OCN and OPN in the CORM‑3 and control group were similar. ALP activity in the CORM‑3‑osteogenic group was increased from day3 and remained significantly higher compared with all other groups on days3,5and7 (P<0.05). Additionally, the results indicated that the optical density value of alizarin red staining in the CORM‑3‑osteogenic group was significantly increased compared with the other groups (P<0.05). Therefore, the present study demonstrated that CORM‑3 may promote the osteogenic differentiation of BMSCs by releasing CO.

CD137 signaling promotes the formation of plaque calcification via inhibiting the fusion of autophagy and lysosomal in Apo E(-/-) mice

Objective: To investigate whether CD137 signaling promoted the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome. Methods: (1) In vivo, CD137 agonist antibody and anti-CD137 antibody were used to stimulate and inhibit the CD137 signaling, respectively. Fifteen Apo E(-/-) mice were randomly divided into three groups: control group (intraperitoneal injection of IgG2b 200 µg) , CD137 agonist group (intraperitoneal injection of CD137 agonist antibody 200 µg) , anti-CD137 group (pretreatment with 200 µg anti-CD137 antibody for 24 hours, then injection of CD137 agonist antibody) . (2) In vitro, primary culture of mouse aortic VSMCs obtained through adherence methods for tissues explants. The cells was divided into three groups: control group, agonist-CD137 group (CD137 agonist antibody 10 μg/ml) , and anti-CD137 group (pretreatment with 10 μg/ml anti-CD137 antibody for 60 minutes, then incubated with 10 μg/ml CD137 agonist antibody) . Von kossa staining was used to detect the calcification in the cell and plaque. Immunohistochemical staining was used to observe the expression of LC3B, Beclin 1 and p62 which are associated with autophagy. The levels of autophagy related protein (LC3) , Beclin 1, p62, and the expression of Runx2 and bone morphogenetic protein 2, which is associated with osteogenic differentiation in the VSMCs, were determined by Western blot. The autophagy flow of each group was detected by fluorescence microscopy. The autophagy was observed by transmission electron microscope in vivo and in vitro. Results: (1) In vivo, the calcified plaque area in CD137 agonist group was significantly larger than that in the control group (3.01%±0.45% vs. 0.27%±0.06%, P<0.01) , and calcified plaque area in anti-CD137 group was significantly smaller compared with that in the CD137 agonist group (1.23%±0.39% vs. 3.01%±0.45%, P<0.05) . Immunohistochemical staining showed that the expression of early autophagy marker protein LC3B and Beclin 1 were significantly upregulated in CD137 agonist group and anti-CD137 group than in control group, and the highest expression was observed in CD137 agonist group (P<0.05) . The expression of advanced autophagy marker protein p62 was higher in the CD137 agonist group than in the anti-CD137 group (P<0.05) . (2) In vitro, the ratio of autophagy related protein LC3 Ⅱ/Ⅰ and p62 protein expression were significantly higher in CD137 agonist group and anti-CD137 group than in control group (P<0.01) , while the expression of p62 protein was significantly higher in CD137 agonist group than that in anti-CD137 group (P<0.05) . In the cell calcification inducing experiment, the expression of BMP-2 and Runx2 protein was significantly higher in CD137 agonist group than that in control group (P<0.01) , but the levels of BMP-2 and Runx2 protein were lower in anti-CD137 group than in CD137 agonist group (P<0.05) . Conclusion: Our results indicate that activation of CD137 signaling can promote the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome.

N-AC-l-Leu-PEI-mediated miR-34a delivery improves osteogenic differentiation under orthodontic force.

Rare therapeutic genes or agents are reported to control orthodontic bone remodeling. MicroRNAs have recently been associated with bone metabolism. Here, we report the in vitro and in vivo effects of miR-34a on osteogenic differentiation under orthodontic force using an N-acetyl-L-leucine-modified polyethylenimine (N-Ac-l-Leu-PEI) carrier. N-Ac-l-Leu-PEI exhibited low cytotoxicity and high miR-34a transfection efficiency in rat bone mineral stem cells and local alveolar bone tissue. After transfection, miR-34a enhanced the osteogenic differentiation of Runx2 and ColI, Runx2 and ColI protein levels, and early osteogenesis function under orthodontic strain in vitro. MiR-34a also enhanced alveolar bone remodeling under orthodontic force in vivo, as evidenced by elevated gene and protein expression, upregulated indices of alveolar bone anabolism, and diminished tooth movement. We determined that the mechanism miR-34a in osteogenesis under orthodontic force may be associated with GSK-3β. These results suggested that miR-34a delivered by N-Ac-l-Leu-PEI could be a potential therapeutic target for orthodontic treatment.

MicroRNA-21 regulates Osteogenic Differentiation of Periodontal Ligament Stem Cells by targeting Smad5

Human periodontal ligament stem cells (hPDLSCs) are mesenchymal stem cells (MSCs) derived from dental and craniofacial tissues that exhibit high potential for differentiation into osteoblasts. Recently, microRNAs (miRNAs) have been established to play important roles in MSC osteogenesis. In the current study, we report that miR-21 was down-regulated in osteogenically differentiated PDLSCs. Overexpression of miR-21 significantly inhibited osteogenesis of hPDLSC, whereas its inhibition demonstrated the opposite effects. Furthermore, SMAD family member 5 (Smad5) was predicted to be a downstream target of miR-21 and was shown to undergo up-regulation in PDLSCs induced toward osteogenesis. Moreover, Smad5 and Runx2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-21. Suppression of miR-21 expression increased the level of Smad5 in vitro and during in vivo transplantation experiments. Furthermore, suppression of Smad5 inhibited osteogenic differentiation and decreased the protein level of Runx2. Taken together, these results suggested that miR-21 be mechanistically implicated in the regulation of osteogenic differentiation of hPDLSCs by targeting Smad5.

Runt-related transcription factor 2 (RUNX2) inhibits apoptosis of intestinal epithelial cells in Crohn’s disease

Apoptosis in intestinal epithelial cells (IECs) prevents the development of Crohn's disease (CD), a type of inflammatory bowel disease (IBD). Runt-related transcription factor 2 (Runx2) inhibits apoptosis in osteosarcoma-derived U2OS cells via down-regulating the transcriptional activity of p53. However, the expression and function of Runx2 in CD remain unclear. In this study, Runx2 protein levels were decreased in the intestinal epithelial cells (IECs) of CD patients and in a mouse 2, 4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model; in contrast, the expression levels of p53 and Bax, a p53-target gene, were increased. In a TNF-α-treated HT29 cell colitis model, the down-regulation of Runx2 was accompanied by the up-regulation of apoptotic markers, including cleaved caspase-3 and Bax. Furthermore, Runx2 overexpression effectively decreased TNF-α-induced Bax and cleaved caspase-3 expression levels. In conclusion, our data indicated that Runx2 might protect IECs from apoptosis in CD, thus revealing a novel molecular target for treating CD.

Down-regulation of Noggin and miR-138 coordinately promote osteogenesis of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can differentiate to osteocytes under suitable conditions. In recent years, micro-nucleotides have been progressively used to modulate gene expression in cells due to the consideration of safety. Our present study aimed to investigate whether co-delivery of Noggin-siRNA and antimiR-138 enhances the osteogenic effect of MSCs. Using a murine MSC line, C3H/10T1/2 cells, the delivery efficiency of Noggin-siRNA and antimiR-138 into MSCs was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Cell phenotype and proliferation capacity was assessed by flow cytometry and MTT assay respectively. The osteogenesis of MSCs was tested by Alkaline Phosphatase (ALP) staining, qRT-PCR, and western blot analyses. Our results demonstrated that the expression of Noggin and miR-138 were significantly silenced in MSCs by Noggin-siRNA and/or antimiR-138 delivery, while the phenotype and proliferation capacity of MSCs were not affected. Down-regulation of Noggin and miR-138 cooperatively promoted osteogenic differentiation of MSCs. The ALP positive cells reached about 83.57 ± 10.18%. Compared with single delivery, the expression of osteogenic related genes, such as Alp, Col-1, Bmp2, Ocn and Runx2, were the highest in cells with co-delivery of the two oligonucleotides. Moreover, the protein level of RUNX2, and the ratios of pSMAD1/5/SMAD1/5 and pERK1/2/ERK1/2 were significantly increased. The activation of Smad, Erk signaling may constitute the underlying mechanism of the enhanced osteogenesis process. Taken together, our study provides a safe strategy for the clinical rehabilitation application of MSCs in skeletal deficiency.

Participation of GATA-3 in regulation of bone healing through transcriptional upregulation of bcl-xL expression

We have previously demonstrated the expression of GATA-DNA-binding protein (GATA)-3, a transcription factor, in osteoblasts and have verified its function in transducing cell survival signaling. This translational study was further designed to evaluate the roles of GATA-3 in regulating bone healing and to explore its possible mechanisms. A metaphyseal bone defect was created in the left femurs of male ICR mice. Analysis by micro-computed topography showed that the bone volume, trabecular bone number and trabecular thickness were augmented and that the trabecular pattern factor decreased. Interestingly, immunohistological analyses showed specific expression of GATA-3 in the defect area. In addition, colocalized expression of GATA-3 and alkaline phosphatase was observed at the wound site. As the fracture healed, the amounts of phosphorylated and non-phosphorylated GATA-3 concurrently increased. Separately, GATA-3 mRNA was induced during bone healing, and, levels of Runx2 mRNA and protein were also increased. The results of confocal microscopy and co-immunoprecipitation showed an association between nuclear GATA-3 and Runx2 in the area of insult. In parallel with fracture healing, Bcl-XL mRNA was significantly triggered. A bioinformatic search revealed the existence of a GATA-3-specific DNA-binding element in the promoter region of the bcl-xL gene. Analysis by chromatin immunoprecipitation assays further demonstrated transactivation activity by which GATA-3 regulated bcl-xL gene expression. Therefore, this study shows that GATA-3 participates in the healing of bone fractures via regulating bcl-xL gene expression, owing to its association with Runx2. In the clinic, GATA-3 may be used as a biomarker for diagnoses/prognoses or as a therapeutic target for bone diseases, such as bone fractures.

The association between parathyroid hormone 1‑34 and the Wnt/β‑catenin signaling pathway in a rat model of osteoarthritis.

The aim of the current study was to determine the effect of parathyroid hormone (PTH) 1–34 on cartilage degeneration, and the association between PTH 1–34 and factors associated with the Wnt/β-catenin pathway following anterior cruciate ligament and medial meniscectomy-induced osteoarthritis (OA) in rats. A total of 64 Sprague-Dawley rats were randomly divided into the following four groups: Sham-operated rats with normal saline (NS)-treatment (n=16); anterior cruciate ligament transection with partial medial meniscectomy (ACLT + MMx) rats with NS-treatment (n=16); sham-operated rats treated with PTH 1–34 (n=16); and ACLT + MMx rats treated with PTH 1–34 (n=16). PTH (15 µg/kg/day) was administered via subcutaneous injection 5 days per week from the first postoperative day for 2 or 6 weeks. Staining with hematoxylin and eosin and safranin O, and a scoring system modified by Mankin were used to assess the histopathological features of cartilage. The present study detected the expression of PTH 1 receptor (PTH1R), sclerostin, dickkopf Wnt signaling pathway inhibitor 1 (DKK1), β-catenin and runt-related transcription factor 2 (RUNX2) in cartilage by immunohistochemical analysis to determine the association between PTH 1–34 and factors associated with the Wnt/β-catenin pathway. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to detect the mRNA expression levels of PTH1R and β-catenin in cartilage. Histological analysis demonstrated that cartilage degeneration was present post-surgery and gradually increased over time. PTH 1–34 reduced the Mankin scores in ACLT + MMx rats compared with the NS-treated ACLT + MMx rats. Immunohistochemistry and RT-qPCR analysis demonstrated that, in cartilage, PTH 1–34 treatment increased the mRNA expression and protein levels of PTH1R and β-catenin, and decreased protein levels of sclerostin, DKK1 and RUNX2 in ACLT + MMx rats compared with the NS-treated ACLT + MMx group. The present study demonstrated that PTH 1–34 upregulated the Wnt/β-catenin signaling pathway and that PTH1-34 downregulated RUNX2 through an alternative pathway to the Wnt/β-catenin signaling pathway, in a rat model of OA.

The role of extracellular signal regulated kinase 1/2 in mediating osteodifferentiation of human periodontal ligament cells induced by cyclic stretch

This study aimed to investigate the mechanism of cyclic stretch that promotesthe osteogenic differentiation of human periodontal ligament cells (hPDLCs) through the mediation of extracellular-signal-regulated kinase 1/2 (ERK1/2). hPDLCs were isolated through the explant method and cultured in vitro. hPDLCs were mechanically stimulated by a multi-channel cell-stress-loading system for 1, 3, 6, 12, and 24 h. The magnitude of stretch was 10% deformation, and the frequency was 0.5 Hz. Nonloaded cells were used as control group. ERK1/2 activation was blocked by U0126, a specific ERK1/2 pathway inhibitor. Additionally, hPDLCs were transfected with adenoviral vector encoding dominant negative ERK1/2 (DN-ERK1/2) to continuouslyinhibit ERK1/2 activation. The mRNA and protein levels of target geneswere detected through real-time polymerase chain reaction and Western blot. Cyclic stretching promoted the expression of ERK1/2, osteocalcin (OCN) mRNA, and bone sialoprotein (BSP) mRNA. The expression of runt-related transcription factor (Runx) 2 protein and mRNA also increased at 3 and 6 h of cyclic stretching. The inhibition of ERK1/2 by U0126 and DN-ERK1/2 suppressed the expressionof Runx2 mRNA, OCN mRNA, BSP mRNA, Runx 2 protein, and p-ERK1/2 protein relative to that in stretched cells without the ERK1/2 inhibitor. ERK1/2 is a critical molecule in the mediation ofthe osteogenic differentiation of hPDLCs under mechanical stimulation. ERK1/2 activation induced the elevation of Runx2 protein levels, which may be involved in the stretch-induced expressions of OCN and BSP.

Zaluzanin C (ZC) induces osteoblast differentiation through regulating of osteogenic genes expressions in early stage of differentiation

Zaluzanin C (ZC) is a sesquiterpene lactones used in herbal medicines. This study examined the effects of ZC on osteoblast differentiation. ZC-induced mRNA expressions levels of osteogenic genes in C3H10T1/2 and MC3T3-E1 cells were determined by RT-PCR and qPCR. ZC regulated the expression of key osteogenic genes in the early stage of differentiation, including distal-less homeobox 5 (Dlx5), DNA-binding protein inhibitor (Id1) and Runt-related transcription factor 2 (Runx2). In addition, ZC increased Runx2 promoter activity, as assessed via a luciferase assay, and Runx2 protein level. These results suggest that ZC may enhance osteoblast differentiation by upregulating the expression of osteogenic genes, especially early stage like as Dlx5, Id1 and Runx2.

Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling

Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated.

The influence of a pulsed magnetic field on the expression of runt-related transcription factor 2 in rats after ovary resection

Objective To observe the influence of a pulsating electromagnetic field (PEMF) on the expression of runt-related transcription factor 2 (Runx2) in ovariectomized rats so as to explore the possibility of using PEMFs to treat osteoporosis. Methods Sixty female Sprague-Dawley rats were randomly divided into four groups of 15-a control group (Sham group), an ovary resection group (OVX group), a resection group treated with alendronate sodium (OVX+ ALN group) and a resection group exposed to a PEMF (PEMFs group). Both ovaries were resected to induce osteoporosis, except in the sham group where the adipose tissues around the ovaries were cut bilaterally without resection. After the operation, the ALN group were given 2 mg/kg of alendronate by gavage daily for 30 days, while the PEMFs group was exposed to a 3.8 mT electromagnetic field pulsing at 8 Hz for 40 min twice daily for 30 days. There was no intervention in the other two groups. The animals were sacrificed using intra-peritoneal injection of sodium pentobarbital and the density of their femurs was measured. The expression of Runx2 protein and Runx2 mRNA were detected using western blotting and quantitative PCR. Results The average bone density, Runx2 protein level and Runx2 mRNA level of the PEMFs group were all significantly higher than those of the OVX group, but there was no significant difference between the PEMFs group and ALN group averages. Conclusion PEMF exposure can upregulate the expression of Runx2 protein and Runx2 mRNA in female rats after ovary resection. This may be one of the mechanisms by which PEMFs treat osteoporosis. Key words: Osteoporosis; Pulsed electromagnetic fields; Runx2 gene

Small molecule T63 suppresses osteoporosis by modulating osteoblast differentiation via BMP and WNT signaling pathways

Osteoporosis results from the imbalance between bone resorption and bone formation, and restoring the normal balance of bone remodeling is highly desirable for identification of better treatment. In this study, using a cell-based high-throughput screening model representing Runt-related transcription factor 2 (RUNX2) transcriptional activity, we identified a novel small-molecular-weight compound, T63, as an efficient up-regulator of osteogenesis. T63 increased the alkaline phosphatase (ALPL) activity and mineralization as well as gene expression of Alpl and other osteogenic marker genes in mouse osteoblasts and mesenchymal stem cell-like cells. Upon induction of osteoblast differentiation, T63 inhibited adipogenic differentiation in the pluripotent mesenchymal cells. Consistently, T63 up-regulated RUNX2 mRNA and protein levels, and knockdown of RUNX2 reduced the osteogenic role of T63. Mechanistically, T63 activated both BMPs and WNT/β-catenin signaling pathways. Inhibition of either signaling pathway with specific inhibitor suppressed T63-induced RUNX2 expression and the osteogenic phenotypes. Moreover, T63 markedly protected against bone mass loss in the ovariectomized and dexamethasone treated rat osteoporosis model. Collectively, our data demonstrate that T63 could be a promising drug candidate and deserves further development for potential therapeutics in osteoporosis.

Tumor necrosis factor-alpha inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated nuclear factor-kappaB signaling pathway.

The majority of experiments show that tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of mesenchymal stem cells and pre-osteoblasts by activated nuclear factor-kappaB (NF-κB) signaling. However, the underlying mechanisms by which NF-κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF-α-activated NF-κB signaling on osteogenic differentiation of pre-osteoblasts. Murine MC3T3-E1 pre-osteoblasts were treated with 10ng/mL of TNF-α. NF-κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF-κB signaling inhibition. EphB4 signaling was activated using ephrinB2-fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription-polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p-NF-κB p65 and NF-κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining. TNF-α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3-E1 cells. Activation of EphB4 signaling by ephrinB2-fc promoted osteogenic differentiation of MC3T3-E1 cells, whereas TNF-α impaired the osteogenic differentiation enhanced by ephrinB2-fc. Pyrrolidine dithiocarbamate blocked the activation of NF-κB signaling induced by TNF-α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF-α. TNF-α inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated NF-κB signaling pathway.

Gold nanoparticles promote osteogenic differentiation of human periodontal ligament stem cells via the p38 MAPK signaling pathway.

Gold nanoparticles (AuNPs) are a promising material for use in regenerative medicine due to their biocompatibility and easy functionalization with biomolecules including growth factors, DNA and peptides. In the present study, transmission electron microscopy indicated that the AuNPs were monodisperse and spherical in shape, with an estimated average diameter of 13nm. And the cellular effects of AuNPs on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and the associated signaling pathways in cell differentiation were investigated based on histochemical analysis of alkaline phosphatase activity and mineralization, quantitative polymerase chain reaction, and western blotting. The results indicated that AuNPs enhanced the differentiation of hPDLSCs into osteoblasts, increasing their osteogenic transcriptional profile including alkaline phosphatase, osterix, collagen type I and runt‑related transcription factor 2 (RUNX2) and activating the p38 mitogen‑activated protein kinase (MAPK) signaling pathway. Furthermore, AuNPs increased the protein level of RUNX2, which is crucial for osteogenic differentiation. These results suggested that AuNPs stimulate the osteogenesis of hPDLSCs partially via activation of the p38 MAPK signaling pathway.

Enamel matrix derivative enhances the proliferation and osteogenic differentiation of human periodontal ligament stem cells on the titanium implant surface

ABSTRACTPeriodontal ligament stem cells (PDLSCs) have mesenchymal-stem-cells-like qualities, and are considered as one of the candidates of future clinical application in periodontal regeneration therapy. Enamel matrix derivative (EMD) is widely used in promoting periodontal regeneration. However, the effects of EMD on the proliferation and osteogenic differentiation of human PDLSCs grown on the Ti implant surface are still no clear. Therefore, this study examined the effects of EMD on human PDLSCs in vitro. Human PDLSCs were isolated from healthy participants, and seeded on the surface of Ti implant disks and stimulated with various concentrations of EMD. Cell proliferation was determined with Cell Counting Kit-8 (CCK-8). The osteogenic differentiation of PDLSCs was evaluated by the measurement of alkaline phosphatase (ALP) activity, Alizarin red staining, and real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The results indicated that EMD at concentrations (5–60 µg/ml) increased the viability and proliferation of PDLSCs. The treatment with 30 and 60 µg/ml of EMD significantly elevated ALP activity, augmented mineralized nodule formation and calcium deposition, and upregulated the mRNA and protein levels of Runx-2 and osteocalcin (OCN) in the PDLSCs grown on the Ti surface. Further investigation found that EMD treatment did not change the protein levels of phosphatidylinositol-3-kinase (PI3K), p-PI3K, Akt and mTOR, but significantly upregulated the phosphorylated levels of Akt and mTOR. Collectively, these results suggest that EMD stimulation can promote the proliferation and osteogenic differentiation of PDLSCs grown on Ti surface, which is possibly associated with the activation of Akt/mTOR signaling pathway.

MC3T3-E1 세포에서 BMP2에 의한 조골세포의 분화에 일주기 유전자 Per1이 미치는 영향

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that play important roles in a variety of cellular functions. Among BMP family members, BMP2 efficiently promotes osteoblast differentiation through Smad-mediated runt-related transcription factor 2 (Runx2) expression. Several recent studies suggest that BMPs are associated with clock genes, in particular Bmal1. Bmal1 protein heterodimerizes with Clock protein and then induces period 1 (Per1) expression. However, the role of Per1 on osteoblast differentiation remains unclear. In this study, we investigated whether Per1 is involved in osteoblast differentiation. MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression were measured using real-time PCR. Interestingly, BMP2 treatment induced Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and β-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is a positive regulator of osteoblast differentiation.

The role of Runx2 in facilitating autophagy in metastatic breast cancer cells.

Breast cancer metastases cause significant patient mortality. During metastases, cancer cells use autophagy, a catabolic process to recycle nutrients via lysosomal degradation, to overcome nutritional stress for their survival. The Runt-related transcription factor, Runx2, promotes cell survival under metabolic stress, and regulates breast cancer progression and bone metastases. Here, we identify that Runx2 enhances autophagy in metastatic breast cancer cells. We defined Runx2 function in cellular autophagy by monitoring microtubule-associated protein light chain (LC3B-II) levels, an autophagy-specific marker. The electron and confocal microscopic analyses were utilized to identify alterations in autophagic vesicles. The Runx2 knockdown cells accumulate LC3B-II protein and autophagic vesicles due to reduced turnover. Interestingly, Runx2 promotes autophagy by enhancing trafficking of LC3B vesicles. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α-tubulin sub-units of microtubules. Inhibiting autophagy decreased cell adhesion and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide molecular insights into the role of autophagy in metastatic cancer cells.

Wnt/β-Catenin Signaling Activates Expression of the Bone-Related Transcription Factor RUNX2 in Select Human Osteosarcoma Cell Types.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/β-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/β-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/β-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of β-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, β-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of β-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3β inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of β-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/β-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.