OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection

OBJECTIVE: To characterize the mutational profile of patients with core-binding factor acute myeloid leukemia (CBF-AML). METHODS: A total of 81 acute myeloid leukemia patients were recruited, which included 36 cases of CBF-AML and 45 cases of cytogenetically normal acute myeloid leukemia (CN-AML) . Mutations of FLT3-ITD, FLT3-TKD, NPM1, c-KIT, NRAS, KRAS, TET2, IDH1/2, RUNX1, DNMT3A, GATA2, ASjXL1, TP53, PTPN11, JAK2V617F, SETBP1 and CEBPA genes were simultaneously detected by DNA-based PCR and Sanger sequencing. RESULTS: Over all, mutations were detected in 68 patients (83.9%), with the most common ones including double CEBPA mutations (n=17), followed by NPM1 (n=15), c-KIT (n=11), NRAS (n=10), TET2 (n=9), FLT3-TKD (n=9), FLT3-ITD (n=8), IDH1 (n=7), RUNX1 (n=7), KRAS (n=7), DNMT3A (n=6), IDH2 (n=4), and GATA2 (n=4) mutations. AML1-ETO and CBFβ-MYH11 fusions were present in 21 and 15 patients, respectively. Coexistence of ≥2 mutations was more common in CN-AML comparing with CBF-AML. The mutation rate of NPM1, FLT3-ITD, DNMT3A, IDH1 and CEBPA double mutations were higher in patients with CN-AML. NRAS, c-KIT and KRAS mutations were identified more frequently in patients with CBF-AML (P<0.05). Based on the function, aberration of genes involved in DNA methylation, NPM1 proteins and transcription predominated in CN-AML, while tyrosine kinase receptor signaling and RAS pathways have predominated in CBF-AML. CONCLUSION The genomic landscape of CBF-AML patients has differed from that of CN-AML patients. Synergy of fusion genes with particular mutations may impact the clinical phenotype and prognosis of patients.
Core Binding Factors ; genetics ; DNA Mutational Analysis ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Prognosis

To study the genic and non-genic regulation of 50 Hz 0.6 mT pulsed electromagnenic fields (PEMF) on rat calvarial osteoblasts (ROB) differentiation.ROBs were achieved by enzyme digestion, and treated with 50 Hz 0.6 mT PEMFs for 1.5 hours after subculture. The alkaline phosphatase (ALP) activity, mRNA transcription of ALP, Runx2 and OSX and protein expression of Runx2 and OSX were detected at 0, 3, 6, 9 and 12 hours after PEMF treatment.The ALP activity at 3 hours after treatment was significantly higher than that in the control(<0.01), while the mRNA transcription of ALP began to increase at 6 hours after treatment. The mRNA transcription of Runx2 increased immediately after treatment and regressed at 6 hours, then increased again. The protein expression of it corresponded but with a little lag. The mRNA transcription of OSX also raised instantly after treatment, then returned to the level of control at 6 hours, and lower than control at 12 hours significantly. The protein expression of it also corresponded but with a bit delay.There are genic regulation for the protein expression of Runx2 and OSX, and non-genic regulation for the ALP activity on the process of 50 Hz 0.6 mT PEMFs prompts ROBs differentiation.
Alkaline Phosphatase ; metabolism ; radiation effects ; Animals ; Cell Differentiation ; genetics ; radiation effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; radiation effects ; Electromagnetic Fields ; Osteoblasts ; chemistry ; radiation effects ; Osteogenesis ; genetics ; radiation effects ; Rats ; Transcription Factors ; metabolism ; radiation effects

No abstract available.
Adult ; Amino Acid Sequence ; Bone Marrow/metabolism/pathology ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Core Binding Factor Alpha 2 Subunit/*genetics ; Exons ; Female ; Humans ; INDEL Mutation ; Leukemia, Myeloid, Acute/*genetics/pathology ; Multiplex Polymerase Chain Reaction ; Proto-Oncogene Proteins/*genetics ; Proto-Oncogene Proteins c-kit/*genetics ; Transcription Factors/*genetics ; *Translocation, Genetic

OBJECTIVEThis work investigated mesenchymal stem cells (MSCs) modified with Runt-related transcription factor 2 (Runx2) therapy for bone regeneration in rabbit mandibular distraction osteogenesis.

METHODSForty-eight New Zealand mature white rabbits were randomly divided into three groups after the rabbit model of mandibular distraction osteogenesis was established: reconstruction plasmid modified with Runx2 (group A), plasmid without Runx2 (group B), and the same dose of saline as control (group C). At the fifth day of distraction phase, MSCs with reconstruction plasmid modified with adv-hRunx2-gfp were injected into the distraction gap of group A. MSCs with reconstruction plasmid modified with adv-gfp was injected into the distraction gap of group B, whereas group C was injected with the same dose of saline. At 8 weeks after injection, all animals were sacrificed, and the distracted mandibles were harvested. The general imaging histological observation and three-point bending test were used for evaluation.

RESULTSCT plain scan and histological analysis confirmed that the amount of new bone forming in the distraction gap of group A was significantly higher than those in groups B and C. Dual-energy X ray and three-point bending test results also showed that the bone mineral density, bone mineral content, and maximum load of the distraction gap of group A were significantly higher than those of groups B and C (P<0.01).

CONCLUSIONRunx2-ex vivo gene therapy based on MSCs can effectively promote the bone regeneration in rabbit mandibular distraction osteogenesis and shorten the stationary phase. Therefore, reconstruction of craniofacial fracture would be a valuable strategy

Absorptiometry, Photon ; Animals ; Bone Density ; Bone Regeneration ; physiology ; Core Binding Factor Alpha 1 Subunit ; genetics ; pharmacology ; Genetic Therapy ; Mandible ; physiology ; surgery ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; Osteogenesis ; genetics ; Osteogenesis, Distraction ; methods ; Plasmids ; Rabbits ; Random Allocation ; Transcription Factors ; genetics ; physiology ; Treatment Outcome

BACKGROUND: To identify potential molecular prognostic markers in core binding factor (CBF) AML, we analyzed incidences and prognostic impacts of mutations in c-KIT, WT1, CEBPA, CBL, and a number of epigenetic genes in CBF AML. METHODS: Seventy one and 21 AML patients with t(8;21) and inv(16) were enrolled in this study, respectively. NPM1, CEBPA, c-KIT, IDH1/2, DNMT3A, EZH2, WT1, and CBL mutations were analyzed by direct sequencing. Patients were categorized with respect to c-KIT and WT1 mutation status, and both clinical features and prognoses were compared. RESULTS: The incidences of FLT3 internal tandem duplication (ITD), NPM1, CEBPA, IDH1/2, DNMT3A, EZH2, and CBL mutations were low (< or =5%) in CBF AML patients. However, c-KIT and WT1 mutations occurred frequently (10.9% and 13.8%, respectively). t(8;21) patients with c-KIT mutations showed significantly shorter overall survival (OS) and disease free survival (DFS) periods than those without mutations (P<0.001, for both); however, although the limited number of t(8;21) patients were analyzed, WT1 mutation status did not affect prognosis significantly. Relapse or death during follow-up occurred more frequently in t(8;21) patients carrying c-KIT mutations than in those without the mutation, although the difference was significant only in a specific patient subgroup with no WT1 mutations (P=0.014). CONCLUSIONS: The incidences of mutations in epigenetic genes are very low in CBF AML; however, c-KIT and WT1 mutations occur more frequently than others. The poor prognostic impact of c-KIT mutation in t(8;21) AML patients only applies in a specific patient subgroup without WT1 mutations. The prognostic impact of WT1 mutation in CBF AML is not evident and further investigation is required.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group/*genetics ; CCAAT-Enhancer-Binding Proteins/*genetics ; Child ; Core Binding Factors/genetics ; Disease-Free Survival ; Epigenesis, Genetic ; Female ; Humans ; Incidence ; Leukemia, Myeloid, Acute/*diagnosis/epidemiology/genetics ; Male ; Middle Aged ; Mutation ; Prognosis ; Proto-Oncogene Proteins c-cbl/*genetics ; Proto-Oncogene Proteins c-kit/*genetics ; Republic of Korea/epidemiology ; Survival Rate ; Translocation, Genetic ; WT1 Proteins/*genetics ; Young Adult

Hedgehog signaling pathway is a conserved and important signaling pathway involved in proliferation and differentiation of many types of cells. Latest studies have found that Hedgehog signaling pathway may induce MSCs osteoblast differentiation by increasing the expression of the Runx2 and Osx and inhibit MSCs differentiate to adipocyte. Hedgehog signaling pathway may also promote osteoblast proliferation by regulating cyclin. This review summarizes the mechanism that Hedgehog signaling pathway regulates osteoblast differentiation and proliferation,and concludes that Hedgehog signaling pathway can regulate bone metabolism. It might provide new ideas for the treatment of osteoporosis.
Cell Differentiation ; Core Binding Factor Alpha 1 Subunit ; genetics ; physiology ; Hedgehog Proteins ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology ; Osteoporosis ; drug therapy ; etiology ; Signal Transduction ; physiology ; Sp7 Transcription Factor ; Transcription Factors ; genetics ; physiology

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.
Ascorbic Acid ; pharmacology ; Butadienes ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Co-Repressor Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Humans ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; Phosphorylation ; drug effects ; RNA Interference ; RNA, Small Interfering ; metabolism ; Stem Cells ; cytology ; Transcription Factors ; antagonists & inhibitors ; genetics ; metabolism ; Up-Regulation ; drug effects

OBJECTIVETo investigate the effect of exposure to static magnetic fields (SMFs) of 3.9 mT on proliferation and differentiation of osteoblasts in vitro.

METHODSThe newborn rat calvarial osteoblasts were isolated by enzyme digestion and randomly divided into 9 groups after one passage. The intensity of the SMFs was 3.9 mT. The cells were exposed in the SMFs for 0 (control group), 0.5, 1.0, 1.5, 2.0, 2.5, 3, 3.5 and 4.0 h groups respectively. They were observed under the contrast phase microscope each day. After 48 h, cell proliferation was assayed by MTT method. The alkaline phosphatase (Alkaline Phosphatase, ALP) activities and calcium content were measured after 3, 6, 9, and 12 days exposed with SMFs. The ALP positive colonies were histochemically stained after 8 days and the calcified nodules were stained by Alizarin Bordeaux after 10 days; BMP-2, Runx-2 and Opg mRNA expression were measured after SMFs treatment in 0, 24, 48 and 72 h.

RESULTSContrast with control group, all SMFs groups enhanced cell proliferation (P < 0.01 or P < 0.05), and they promoted maturation and mineralization of the osteoblasts. The results showed that SMFs improved the ALP activity, promoted calcium content, boost BMP-2, Runx -2 and Opg mRNA expression.

CONCLUSIONThe cells exposed to the SMFs of 3.9 mT at 2.5 h apparently promote proliferation and differentiation of osteoblasts in vitro.

Animals ; Bone Morphogenetic Protein 2 ; genetics ; Calcium ; metabolism ; Cell Differentiation ; radiation effects ; Cell Proliferation ; radiation effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; Magnetic Fields ; Osteoblasts ; physiology ; radiation effects ; Osteoprotegerin ; genetics ; Rats ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo analyze the effects of calcium hydroxide [Ca(OH)2] on transcription of the bone sialoprotein (BSP) gene in human dental pulp cells.

METHODSHuman dental pulp tissues were collected from extracted teeth for orthodontic reason. In cell culture media, different dose (0.012, 0.120, 0.400 and 1.200 mmol/L) of Ca(OH)2 was added. Total RNA of cells were extracted. The best dose of Ca(OH)2 on human BSP was determined with the real-time polymerase chain reaction (PCR). Further, the time (0, 3, 6, 12, 24 h) effects of the best dose Ca(OH)2 on human BSP, runt-related transcription factor-2 (Runx-2) and osterix (OSX) mRNA levels were determined with PCR. Further method included transient transfection assays, linking chimeric constructs of the human BSP gene promoter to a luciferase reporter gene, then ransfected using lipofectamine in cells and measured the luciferase activities of BSP gene promoter.

RESULTSWith the real-time PCR, the optimal Ca(OH)2 concentration was determined as 1.200 mmol/L. With this concentration at different time points (0, 3, 6, 12 and 24 h), the levels of BSP mRNA increased at 6 h (1.45 ± 0.36), reached maximal at 12 h (2.66 ± 0.18); the levels of Runx-2mRNA increased at 6 h (2.38 ± 0.08), at 12 h (2.73 ± 0.16), and decreased at 24 h. OSX mRNA could be recognized at 12 h, reached maximal levels at 24 h (3.30 ± 0.062). Transient transfection assays showed that treatment of human dental pulp cells with Ca(OH)2 (1.200 mmol/L) increased the luciferase activities of the constructs between -84LUC and -868LUC at 12 h (2.00 ~ 2.60 fold).

CONCLUSIONSThis study demonstrate that Ca(OH)2 could stimulate BSP transcription between -84LUC and -868LUC in the human BSP gene promoter in human dental pulp cells.

Calcium Hydroxide ; pharmacology ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Dental Pulp ; cytology ; metabolism ; Humans ; Integrin-Binding Sialoprotein ; genetics ; metabolism ; Luciferases ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Sp7 Transcription Factor ; Transcription Factors ; genetics ; metabolism ; Transcription, Genetic ; drug effects ; Transfection