mRNA Expression Of Runx2 Research Articles

Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

ABSTRACTAim of the study: The peripheral nervous system is involved in regulation of bone metabolism via sensory and sympathetic innervation. Substance P (SP) and calcitonin gene-related peptide (CGRP) are two sensory neuropeptides that have been associated with regulation of osteogenic differentiation. However, the interaction between SP and CGRP both with each other and the bone morphogenetic protein 2 (BMP2) in regulation of osteogenic differentiation has not been studied. Therefore, the aim of this study was to investigate the interaction between SP and CGRP on BMP2-induced bone differentiation using model progenitor cells. Materials and methods: C2C12 myoblasts and MC3T3 pre-osteoblasts were treated with SP and CGRP, both individually and in combination, in the presence of BMP2. The effects of the neuropeptides on BMP2-induced osteogenic differentiation were assessed by measuring alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic markers. Results: Both SP and CGRP enhanced BMP2 signaling, Runx2 mRNA expression, as well as mineralization in vitro. Co-stimulation with SP and CGRP resulted in down-regulation of BMP2-induced bone differentiation, suggesting potential crosstalk between the two neuropeptides in regulation of BMP2 signaling. Conclusions: Based on the results shown here, CGRP can mitigate augmenting effects of SP on BMP2 signaling and the three pathways potentially converge on Runx2 to regulate BMP2-induced bone differentiation.

Abstract 660: Elevated Calcium Drives Inflammatory-Driven Angiogenesis

Background: Peripheral arterial disease (PAD) is characterised by accelerated arterial calcification and impairment in angiogenesis, both of which can influence cardiovascular clinical outcomes. Studies implicate calcification as a driver of PAD, however, the mechanisms by which calcification modulates angiogenesis remain poorly understood. This study assessed the effect of high calcium on angiogenesis both in vitro and in vivo . Methods: Human Coronary Artery Endothelial Cells (EC) were cultured and treated with calcification medium (CM) (CaCl 2 2.7mM, Na 2 PO 4 2.0mM) for 24 h. Angiogenic assays of proliferation, migration and tubulogenesis were conducted, and immunoblotting assessed angiogenic regulatory proteins. In vivo studies employed a calcification model with 8-12-week-old male OPG -/- and wildtype (C57BL6/J, control) mice which underwent hind-limb ischaemia (HLI) surgery. Blood flow reperfusion was assessed by Laser Doppler Perfusion Imaging (LDPI). Calcium assay assessed calcium levels in blood serum of the C57BL6/J and OPG -/- mice. Results: CM significantly reduced EC tubulogenesis and viability (34% and 58% p<0.05) but increased migration (p<0.0001) over 24h. There was a significant increase in the protein levels of angiogenic regulators VEGFA (p<0.0001) and HIF-1α (p<0.0001). CM also significantly increased NF-κB (P65) nuclear protein, a key mediator of inflammatory-driven angiogenesis. qRT-PCR showed upregulation of osteoinductive factor Bone Morphogenetic protein (BMP2), and transcription factor Runx2 mRNA expression, both involved in osteogenesis and calcification. Blood serum calcium levels were significantly increased in OPG deficient mice. LDPI found there was significantly reduced blood-flow reperfusion in OPG -/- mice at days 6 (0.03±0.01 vs 0.32±0.05 p<0.01) and day 10 (0.08±0.01 vs 0.35±0.05 p<0.01) post HLI induction, compared to controls. Conclusion: This is the first demonstration that high-levels of calcium impair ischaemia-driven angiogenesis in vivo and cause inflammation in ECs that suppresses tubule formation in vitro , despite upregulation of key angiogenic regulators VEGFA and HIF-1α. These findings have implications for the development of therapies that can suppress calcification in PAD.

Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells

This study aimed to construct the expression of bone morphogenetic protein-4 (BMP4) lentiviral vector gene and explore its influence on the biological activity of mouse induced pluripotent stem (iPS) cells. iPS cell lines stably overexpressing BMP4 were constructed by lentivirus transfection (BMP4-overexpressing group). Cells without transfection served as the blank group, and cells with only vector transfection served as the empty-vector group. Cell proliferation was detected by CCK8, and the expression levels of ameloblastin (AMBN), cytokeratin (CK) 14, dentin sialophospho-protein (DSPP), bone sialoprotein (BSP), and Runx2 mRNA were detected by quantitative polymerase chain reaction. Alkaline phosphatase (ALP) activity was used to detect the degree of cell differentiation. Compared with blank and empty-vector groups, proliferation activity and ALP activity of BMP4-overexpressing group obvious increased (P<0.05), BMP4, AMBN, CK14, DSPP, BSP, Runx2 mRNA expression also increased (P<0.05). BMP4 can significantly promote the odontogenic differentiation of iPS.

Fate of adipose derived stem cells is correalted with expression of specific set of genes

Purpose: Adipose tissue represents an abundant, accessible and rich source of adult stem cells with the ability to differentiate into multiple lineages. Our understanding of the dynamic process requiring indispensable differentiation of adipose-derived stem cells (ASCs) remains limited. Here, we describe that differentiation of ASCs into adipogenic, osteogenic, and chondrogenic lineages is dependent on expression of certain specific genes. Methods: ASCs were isolated from human adipose tissue by enzymatic digestion using collagenase. Isolated ASCs were cultured as monolayer in DMEM low-glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37°C in a 5% CO2 incubator. Adipogenic differentiation of ASCs was done for 14 days using adipogenic medium. Osteogenic and chondrogenic differentiation of ASCs was done for 21 days using osteogenic and chondrogenic differentiation medium. Adipogenic differentiation of ASCs was accessed by Oil Red O staining, and gene expression of adipogenic markers. Osteogenic differentiation was accessed by Von Kossa staining, calcium estimation and gene expression of osteogenic markers. Chondrogenic differentiation was accessed by Alcian blue staining, and gene expression of chondrogenic markers. Results: Normal differentiation of ASCs into adipogenic lineage resulted in higher level of oil red o staining which represents higher adipose depots. The mRNA expression of adipogenic markers Adiponectin, FABP4, and PPAR-γ significantly increases with adipogenic differentiation. Interestingly, expression of TAZ decreases with adipogenic differentiation of ASCs. Differentiation of ASCs into osteogenic lineage resulted in higher amount of von Kossa staining along with significantly higher level of calcium concentrations. The mRNA expression of osteogenic markers RUNX2, Osteonectin, Osteopontin, ALP, and TAZ significantly increases during osteogenic differentiation. Differentiation of ASCs into chondrogenic lineage resulted in higher level of Alcian blue staining. The mRNA expression of chondrogenic markers Aggrecan, COL2A1, SOX9, and TAZ significantly increases during chondrogenic differentiation. Conclusions: We demonstrate that differential gene expression has critical role in ASCs lineage determination. By identifying and modulating expression of such genes, we may direct the differentiation fate of ASCs that will be useful as a cell therapy for regenerative medicine.

Exendin-4 relieves the inhibitory effects of high glucose on the proliferation and osteoblastic differentiation of periodontal ligament stem cells

BackgroundWith the impaired regenerative potential in patients with diabetes mellitus (DM), Periodontal ligament stem cells (PDLSCs) are regarded as an attractive source of stem cells for periodontal cytotherapy. Recent studies have shown that Exendin-4 (Ex-4) exerts cell-protective effects and bone remodeling ability on many types of cells. The aim of this study was to investigate whether Ex-4 alleviates the inhibition of high glucose on the proliferation and osteogenic differentiation of PDLSCs. MethodsPDLSCs were incubated in medium supplemented with 5.5 mM d-glucose (NG), 30 mM d-glucose (HG), NG plus Ex-4, and HG plus different concentration (1, 10, 20, 100 nM) of Ex-4 respectively. Cell proliferation was detected by CCK-8 assay and cell cycle analysis. Osteogenesis was assessed by Alizarin Red S staining and evaluation of the mRNA expression of Runx2, ALP and Osx at day 7, 14 and 21. Intracellular level of reactive oxygen species (ROS) was detected using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CMH2DCF-DA). ResultsThe proliferation ability, mineralized nodules forming capacity and the mRNA expression of Runx2, ALP and Osx of PDLSCs in HG group were decreased, the ROS level was increased compared to NG group. With the treatment of Ex-4, the HG-inhibited proliferation ability and osteogenic differentiation ability of PDLSCs were significantly reversed, the HG-increased ROS level could be down-regulated. Moreover, Ex-4 enhanced the osteogenic differentiation of normal PDLSCs. ConclusionsEx-4 alleviates the inhibitory effect of HG on the proliferation and osteoblastic differentiation of PDLSCs, and has a significant enhance in the osteoblastic differentiation of normal PDLSCs, giving new insights into the possible therapeutic method of diabetic periodontitis.

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.

Molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rat bone marrow stromal stem cells (rBMSCs)

ObjectiveThe study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rBMSCs. MethodsrBMSCs were harvested from SD rats, and determination of alkaline phosphatase (ALP) activity, quantification of mineralization by Alizarin Red S staining, and the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) by RT-PCR after rBMSCs stimulated by osteogenic induction with (0.1–10) µg/mL of quercitrin, quantification of Lipid droplet by Oil Red O staining and the mRNA expression of adipogenic differentiation marker (PPARγ, C/EBPα, and aP2) by RT-PCR after rBMSCs stimulated by adipogenic induction with (0.1-10) µg/mL of quercitrin. ResultsQuercitrin can up-regulate the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) and increase ALP activity and mineralization after osteogenic induction, on the other hand quercitrin can suppress the mRNA expression of adipogenic differentiation markers (PPARγ, C/EBPα, and aP2) and decrease lipid droplet after adipogenic induction. ConclusionThis study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of rBMSCs, which was associated with the up-regulation of Runx2, BMP-2, and OSX mRNA expression and the down-regulation of PPARγ, C/EBPα, and aP2 mRNA expression.

Effect of miR-21 down-regulated by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells

To explore the effect and mechanism of miR-21 down-regulated which was induced by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 μmol/L) of H 2O 2. The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H 2O 2 was obtained. To analyze the effect of H 2O 2 on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H 2O 2 group (group B), osteogenic induction group (group C), and H 2O 2+osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H 2O 2 group (group A1), H 2O 2+osteogenic induction group (group B1), H 2O 2+osteogenic induction+miR-21 inhibitor group (group C1), and H 2O 2+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H 2O 2 group (group A2), H 2O 2+osteogenic induction group (group B2), H 2O 2+osteogenic induction+siRNA-PTEN negative control group (group C2), and H 2O 2+osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining. The results of MTS and RT-PCR showed that the appropriate concentration of H 2O 2 was 160 μmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks ( P<0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D ( P<0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 ( P<0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks ( P<0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks ( P<0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week ( P<0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2. H 2O 2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.

MicroRNA-214-5p/TGF-β/Smad2 signaling alters adipogenic differentiation of bone marrow stem cells in postmenopausal osteoporosis

Postmenopausal osteoporosis (OPM) is a common type of osteoporosis in females. It is a systemic, chronic bone disease that presents as microstructure degradation of osseous tissue, decreased bone mineral density and increased osteopsathyrosis caused by hypoovarianism and reduced estrogen levels in the body following menopause. In the present study, the role of microRNA (miR)-214-5p in the regulation of the expression of bone marrow stem cells (BMSCs) was investigated, and its molecular mechanism of osteogenic induction in vitro was assessed. When dexamethasone-induced adipogenic differentiation was performed, miR-214-5p expression was increased compared with the control group, as determined by RT-qPCR. Furthermore, oil red O staining, RT-qPCR and western blot analysis demonstrated that overexpression of miR-214-5p promoted adipogenic differentiation, inhibited alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OC) and collagen α-1 (I) chain (COL1A1) mRNA expression, and suppressed transforming growth factor (TGF)-β, phosphorylated (p)-Smad2 and collagen type IV α1 chain (COL4A1) protein expression in BMSCs. Additionally, downregulation of miR-214-5p increased the ALP, Runx2, OC and COL1 mRNA expression and increased TGF-β, Smad2 and COL4A1 protein expression in BMSCs. Furthermore, a TGF-β inhibitor was employed to inhibit TGF-β expression in BMSCs following miR-214-5p downregulation, which led to reduced Smad2, TGF-β and COL4A1 protein expression, and ALP, Runx2, OC and COL1 mRNA expression was also reduced, compared with the miR-214-5p downregulation only group. It was demonstrated that miR-214-5p may weaken osteogenic differentiation of BMSCs through regulating COL4A1. In conclusion, the results of the present study indicated that miR-214-5p may promote the adipogenic differentiation of BMSCs through regulation of the TGF-β/Smad2/COL4A1 signaling pathway, and potentially may be used to develop a novel drug for postmenopausal osteoporosis.

High-power, red-light-emitting diode irradiation enhances proliferation, osteogenic differentiation, and mineralization of human periodontal ligament stem cells via ERK signaling pathway.

Light-emitting diode (LED) is attracting attention as a new light source for phototherapy. However, its effects on periodontal tissue regeneration remain unknown. The aim of this study was to examine the effects of high-power, red LED irradiation on human periodontal ligament stem cells (PDLSCs), which play an important role in periodontal tissue regeneration. PDLSCs were derived from adult human third molars. The light source was red LED (peak wavelength: 650nm). Energy densities ranging from 0 to 10 J/cm2 were tested to determine the optimal dose. PDLSC proliferation was measured using two parameters: live cell protease and ATP levels. After the cells were induced to differentiate, the effect of LED irradiation on osteogenic differentiation and mineralization was examined, with particular focus on the extracellular signal-regulated kinase (ERK)1/2 signaling pathway using an ERK inhibitor (PD98059). LED irradiation at 8 J/cm2 led to a significant increase in PDLSC proliferation and enhanced Runx2 and Osterix mRNA expression, Alkaline phosphatase activity, procollagen type I C-peptide and osteocalcin production, calcium deposition, and alizarin red S staining. In addition, LED induced the activation of ERK1/2, and the effects of LED on PDLSC proliferation, differentiation, and mineralization could be suppressed by treatment with PD98059. The results of this study show that 650-nm high-power, red, LED irradiation increases PDLSCs proliferation, and osteogenic differentiation and mineralization, mediated by ERK1/2 activation. These findings suggest that LED may be a useful tool for periodontal tissue regeneration.

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.

The role of glutathione in steroid induced bone marrow mesenchymal stem cells dysfunction

To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs). The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall method in vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×10 5 cells/mL); group B, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot. The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance ( A) values had significant differences between groups ( P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E ( P<0.05), but there was no significant difference among groups A, D, and E ( P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups ( P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups ( P<0.05). GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.

Klf10 silencing inhibited mechanical force induced osteogenic differentiation of human periodontal ligament cells

To investigate the effect of Klf10 silence on human periodontal ligament cells (HPDLCs) under mechanical force. HPDLCs were isolated and transfected with Klf10 siRNA, and then exposed to centrifugal force for 6 h at 631 r/min. Purmorphamine, an hedgehog signaling pathway agonist, was used for intervention. The activity of alkaline phosphatase (ALP) was detected by ELISA. RT-PCR and Western blot were performed to detect the mRNA and protein expression of Klf10, Runt related transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN). The protein expression of glioma associated oncogene homolog 1 (Gli1) and patched-1 (PTCH1) was detected by Western blot. SPSS 20.0 software package was used to analyze the data. Mechanical force increased mRNA and protein level of Klf10, Runx2, OPN and OCN, and elevated ALP activity significantly (P<0.05). Mechanical force also upregulated the protein expression of GLI1 and Ptch1 significantly (P<0.05). Compared with the control group, Klf10 siRNA transfection significantly decreased mRNA and protein level of Klf10 (P<0.05). Klf10 siRNA significantly inhibited the activity of ALP, and downregulated mRNA and protein expression of Runx2, OPN and OCN (P<0.05). Moreover, Klf10 siRNA significantly inhibited protein expression of GLI1 and Ptch1 (P<0.05), and purmorphamine obviously inhibited the effect of Klf10 siRNA (P<0.05). Klf10 silencing could inhibit bone differentiation of human periodontal ligament cells under mechanical force, which may be through regulation of hedgehog signaling pathway.

Anti-osteoporotic activities of the collagen extracted from Carapacis et Plastri Testudinis on a ROS1728 cell line in vitro

Purpose: The decoction of collagen extracted from Carapacis et Plastri Testudinis (CCPT) has been reported to have an effect of anti-osteoporosis. The present study was to investigate the in vitro molecular mechanisms effect of CCPT on ROS1728 differentiation and proliferation. Methods: Rat osteoblast cell line (ROS 1728) was cultured and exposed to CCPT at different concentrations. The effect of CCPT on the proliferation of ROS1728 was evaluated through detecting the activity of Alkaline Phosphatase (ALP) and the type I collagen (Coll-I) content. The gene expression of ALP, Coll-I, Osteocalcin (OC), Runx2, Osterix (Osx), Osteoprotegerin (OPG) and RANKL (receptor activator of NF-MB ligand) was observed using RT-PCR analysis. Results: The results showed that CCPT treatment (2.5, 5.0 and 10.0 mg/ml) affected the proliferation of ROS1728 in a concentration-dependent manner. The highest expression was obtained by treatment with 5 mg/ml of CCPT after a nine-day treatment duration based on ALP activity and Coll-I content (P<0.01) with the control group, whereas, the expression of ALP, Coll-I, OC, Runx2, Osx and OPG mRNA was facilitated (P<0.01). Only the level of RANKL mRNA decreased compared with the control group (P<0.01). Conclusion: Use of CCPT can induce ROS1728 proliferation into osteoblasts and enhance the function of osteogenesis through regulating the gene expression of Runx2 and the osteogenesis-specific transcription factors of Osx, and inhibit bone absorption by OPG/RANKL/RANK signaling pathways.

GOLM1 Stimulation of Glutamine Metabolism Promotes Osteoporosis via Inhibiting Osteogenic Differentiation of BMSCs

Background/Aims: Bone marrow mesenchymal stem cells (BMSCs) play an essential role in osteoporosis. However, the molecular mechanisms and the involvement of glutamine metabolism in osteogenic BMSCs differentiation and osteoporosis remain largely unclear. In this study, we investigated the role of Golgi membrane protein 1 (GOLM1) and glutamine metabolism in BMSCs differentiation and osteoporosis. Methods: Osteogenic differentiation-inducing media (Odi) was used to induce the osteogenic differentiation of BMSCs. The mRNA expression of GOLM1, ALP, Runx2, Osx, BSP and OCN was determined by qRT-PCR assay. Western blot assay was used to analyze GOLM1, p-mTOR, mTOR, p-S6 and S6 abundance in GOLM1 silencing and over-expressed BMSCs. Glutamine uptake, intracellular glutamine, glutamate and α-KG level was detected using indicated Kits. GOLM1 antibody, glutamine metabolism inhibitors EGCG and BPTES were used to treat ovariectomy (OVX)-induced osteoporosis. Bone mineral density and bone volume relative to tissue volume (%) were analyzed by micro-CT. Serum was collected from osteoporosis patients and healthy participants and subjected to GOLM1 determination using ELISA Kit. Results: GOLM1 expression and glutamine metabolism were suppressed by Odi. GOLM1 blockage or inhibition of glutamine metabolism promoted the osteogenic differentiation of BMSCs induced by Odi. GOLM1 activated glutamine metabolism depending on the mTOR signaling pathway. In vivo, GOLM1 antibody or combination of glutamine inhibitor EGCG and BPTES rescued the osteoporosis in an OVX-operated mouse model. Serum GOLM1 level was increased in the patients of osteoporosis compared with healthy people. Conclusion: GOLM1 stimulates glutamine metabolism to suppress the osteogenic differentiation of BMSCs and to promote osteoporosis. Therefore, GOLM1 activation of glutamine metabolism is a potential target for osteoporosis.

Isolation and screening of rare Actinobacteria, a new insight for finding natural products with antivascular calcification activity.

Vascular calcification (VC) is a significant pathological process in some life-threatening diseases. Several pathological mechanisms, including transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and apoptosis are involved in VC. Compounds with an inhibitory effect on these processes are potentially efficient medications. In consideration of the multiple biological activities of Actinobacteria, this research was aimed at finding anti-VC metabolite-producing Actinobacteria. After the isolation and identification of Actinobacteria, the effect of their fermentation broth extracts on the apoptosis rate was measured using various methods, for example, ethidium bromide/acridine orange staining, DNA laddering and diphenylamine assays. The effect of the most effective fermentation broth extract of Actinobacteria (FBEA) on the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) was examined. Finally, the most effective FBEA was fractionated and the chemical composition of anti-VC fractions was analysed using GC-MS. Various VC inhibition rates were observed in the tested FBEA (20μgml-1 ; 17·9-60·15%). The inhibition of DNA fragmentation was 7-48%. The FBE with the greatest anticalcification activity belonged to Kribbella sp. UTMC 267 and, according to 16S rRNA analysis, Kribbella sancticallisti with a similarity of 98·53% is its nearest neighbour. The FBE of Kribbella sp. UTMC 267 reduced Runx2 mRNA expression by 2·95-fold and OPN mRNA expression by 28·57-fold, both of which are considered significant (P<0·05). Finally, GC-MS analysis showed the existence of potent anti-oxidative and anti-inflammation agents in FBE of Kribbella sp. UTMC 267. Actinobacterial metabolites can provide a new strategy for treating VC diseases by reducing the expression of osteogenic genes, the apoptosis rate and oxidative stress. This study highlights the therapeutic potential of Kribbella sp. metabolites and Actinobacteria as a new natural source for drug discovery programs in the nonantibiotic bioactivity field.

Inhibition of adenosine monophosphate-activated protein kinase suppresses bone morphogenetic protein-2-induced mineralization of osteoblasts via Smad-independent mechanisms.

Previous studies showed that adenosine monophosphate-activated protein kinase (AMPK), which plays as an intracellular energy sensor, promotes the differentiation and mineralization of osteoblasts via enhancing expression of bone morphogenetic protein (BMP)-2, which is a potent inducer of osteoblastogenesis. Thus, the aim of this study was to examine the roles of AMPK in BMP-2-induced osteoblastogenesis. We used a murine osteoblastic cell line MC3T3-E1 and a murine marrow stromal cell line ST2. BMP-2 (50 and 100 ng/mL) stimulated alkaline phosphatase (ALP) activity and enhanced mineralization of MC3T3-E1 cells, while the effects of BMP-2 were partly abolished by an inhibitor of AMPK, ara-A (0.1 mM). Real-time PCR showed that BMP-2 significantly increased the mRNA expressions of Alp, osteocalcin (Ocn), Runx2, Osterix and Dlx-5 in MC3T3-E1 cells, while co-incubation of ara-A significantly decreased the BMP-2-stimulated expression of Alp, Ocn, and Runx2. Moreover, co-incubation of ara-A suppressed the BMP-2-induced upregulation of Alp and Ocn in ST2 cells. Western blot analysis showed that BMP-2 phosphorylated Smad1/5 although it did not affect AMPK phosphorylation in MC3T3-E1 cells. Furthermore, a BMP receptor inhibitor LDN-193189 inhibited the phosphorylation of Smad1/5, but did not affect AMPK. In addition, co-incubation of ara-A did not affect BMP-2-induced phosphorylation of Smad1/5. These findings suggest that the inhibition of AMPK activation reduces the osteo-inductive effects of BMP-2 by decreasing the expression of Alp, Ocn, and Runx2 through Smad-independent mechanisms in osteoblastic cells.

Clinical significance and expression of runt-related transcription factor 2 in esophageal squamous cell carcinoma

Objective To evaluate the expression and clinical significance of runt-related transcription factor (RUNX2) in esophageal squamous cell carcinoma (ESCC). Methods We analzyed the RNA-seq data of esophageal carcinoma patients from The Cancer Genome Atlas (TCGA) database. Quantitative real-time PCR and immunohistochemistry were used to detect RUNX2 mRNA and protein expression in 80 ESCC patients’ cancer and adjacent normal esophageal mucosal tissues. The relationship of RUNX2 protein expression and clinicopathological parameters and the association of RUNX2 protein expression with prognosis were analyzed. Results The expression of RUNX2 mRNA in esophageal carcinoma and esophageal squamous cell carcinoma was significantly increased by TCGA database analysis, P=0.000, P=0.002. The RUNX2 mRNA (P=0.000) and protein (16.942, P=0.000) expression was significantly higher in ESCC cancer tissues than in normal tissues. RUNX2 protein expression was significantly associated with the tumor invasion (6.138, P=0.013), differentiation (6.277, P=0.014) and TNM stage (3.903, P=0.048). The overall survival (P=0.048) and progression free survival (P=0.025) in patients with high RUNX2 protein expression were significantly longer than those in the patients with low RUNX2 protein expression. Multivariate analysis showed that the expression of RUNX2 was an independent risk factor of overall survival (P=0.000) and progress free survival (P=0.002). Conclusion RUNX2 might play an important role in the development and progression of ESCC and might be a prognostic factor in ESCC. Key words: Esophageal squamous cell carcinoma; Runt-related transcription factor 2; Prognosis

Collagen-derived dipeptide prolyl-hydroxyproline promotes osteogenic differentiation through Foxg1

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.

The inhibitory effect of Aconiti Sinomontani Radix extracts on the proliferation and migration of human synovial fibroblast cell line SW982

Ethnopharmacological relevanceAconiti Sinomontani Radix is frequently used in the treatment of Bi syndrome in traditional Chinese medicine. Several reports indicate that Aconiti Sinomontani Radix has therapeutic effects for rheumatoid arthritis (RA). However, the cellular mode of action is still unclear. To investigate the effect of alkaloid extracts of Aconiti Sinomontani Radix on proliferation and migration of human synovial sarcoma SW982 cells as well as the molecular mechanism underlying. Materials and methodsSW982 cells were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Wound scratch assays were performed to assess the migrated rate of SW982 cells. Quantitative real-time PCR was used to measure the mRNA expression levels of Wnt5a, Runx2, MMP3, and Bmp2. Western blotting was used to measure the phosphorylated levels of JNK and NF-κB as well as the expression of MMP3. ResultsThe alkaloid extract from Aconiti Sinomontani Radix (MQA) and MQB, which removed lappaconitine from MQA significantly inhibited the proliferation of SW982 in a dose-dependent manner. The proliferation inhibitory effect of MQB was more potent. Incubation with 10μg/ml MQB for 12, 24, and 36h inhibited the migration of SW982 cells by 83%, 58%, and 42%, respectively. Treatment with different concentrations of MQB for 24h inhibited mRNA expression of Wnt5a, Runx2, and MMP3, but Bmp2 mRNA expression was elevated by MQB. Further, MQB inhibited phosphorylation of JNK and NF-κB p65 as well as MMP3 expression by Western blotting analysis. ConclusionThe results showed that MQB inhibited proliferation and migration of SW982 cells possibly through suppressing Wnt5a-mediated JNK and NF-κB pathways. These results indicated that MQB might be an active extract of Aconiti Sinomontani Radix for targeting fibroblast-like synoviocytes (FLS) and be potential for RA therapy.