CCAAT-Enhancer-Binding Proteins ; genetics ; Cytogenetic Analysis ; Cytogenetics ; methods ; Extremities ; Humans ; Liposarcoma ; etiology ; genetics ; Liposarcoma, Myxoid ; etiology ; genetics ; Molecular Biology ; methods ; Oncogene Proteins, Fusion ; genetics ; RNA-Binding Protein FUS ; genetics ; Retroperitoneal Neoplasms ; etiology ; genetics ; Ring Chromosomes ; Soft Tissue Neoplasms ; etiology ; genetics ; Transcription Factor CHOP ; Translocation, Genetic

OBJECTIVES: Endoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated. METHODS: In primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptasepolymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved. RESULTS: Tunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B. CONCLUSION.: These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.
Activating Transcription Factor 6 ; Carrier Proteins ; CCAAT-Enhancer-Binding Proteins ; Endoplasmic Reticulum Stress ; Endoplasmic Reticulum ; Epithelial Cells ; Glycoproteins ; Humans ; Immunoenzyme Techniques ; Inositol ; Mucins ; Phosphotransferases ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA, Small Interfering ; Transcription Factor CHOP ; Transcription Factors ; Transfection ; Tunicamycin

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.
CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Gene Expression Regulation, Leukemic ; drug effects ; Genes, Regulator ; drug effects ; Humans ; Interferon Regulatory Factor-1 ; metabolism ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; Leukemia, Promyelocytic, Acute ; genetics ; STAT2 Transcription Factor ; metabolism ; Signal Transduction ; Tretinoin ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of metformin on 3T3-L1 preadipocyte's differentiation and consequently observe the anti-proliferative effects of metformin-treated adipocytes on leukemia cells.

METHODSDifferent concentrations of metformin were added in 3T3-L1 preadipocytes to induce maturation, the matured adipocytes were detected by oil red O staining and quantified by absorbance value (OD). Real-time PCR was used to detect the mRNA expression level of the key adipogenic genes PPARγ, C/EBPα and FABP4(ap2). The adipocytes were co-cultured with GFP+-THP-1 cells, 1 µg/ml of cytarabine(Ara-C) was added and incubated for 48 hours, the flow cytometry was used to detect the apoptosis rate of GFP+-THP-1 cells. Adipocyte supernatant was collected and mixed with equal volume of tumor lysat medium (RPMI 1640) at 1:1 to culture tumor cells. The leukemia cell proliferation activity was detected by CCK-8; after 48 hours of adding 1 µg/ml Ara-C, the protective effect on chemotherapy was assayed by using cytometer.

RESULTSThe metformin lowered the OD value, and the expression levels of both adipogenic genes C/EBPα and FABP4 were lower than those of controls, while the expression level of PPARγ mRNA was not significantly changed, the apoptosis rate of leukemia cells co-caltured with metformin-treated adipocytes was higher than that of co-cultured cells without metformin treatment. The adipocytes promoted the leukimia cell proliferation and protected leukemia cells from chemotherapy, which could be abrogated by metformin.

CONCLUSIONThe metformin can inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes, and can regulate the protective effect of adipocytes on the apoptosis, proliferation and chemotherapy of leukemia cells.

3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Animals ; CCAAT-Enhancer-Binding Protein-alpha ; Cell Differentiation ; Cell Proliferation ; Cytarabine ; Drug Resistance, Neoplasm ; Fatty Acid-Binding Proteins ; Humans ; Leukemia ; Metformin ; Mice ; PPAR gamma ; RNA, Messenger

OBJECTIVETo compare the adipogenesis and the adipocyte function between 3T3-L1 cells and human bone marrow mesenchymal stem cells (MSCs) in vitro.

METHODSBy density gradient centrifugation and adherent culture, the MSCs were isolated from human bone marrow and purified. The cell morphology was observed under an inverted microscope. After the induction of adipogenic differentiation, the differentiation level was detected by oil red O staining and OD values. The expression of PPARγ, FABP4 and C/EBPα mRNA was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Adipocytes and THP-1 cells were co-cultured by adding 1 µg/ml cytarabine. The ability of chemotherapy resistance was measured after 48 h.

RESULTSThe Oil Red O staining and measuring the absorbance showed that the lipid content in 3T3-L1 cells group was more than that in MSCs group, and the OD value was higher than that in MSCs group (P < 0.05). The results of qRT-PCR showed that the relative expression of PPARγ, FABP4 and C/EBP&alpha; mRNA of 3T3-L1-derived adipocytes was higher than that of human bone marrow MSCs-derived adipocytes (P < 0.05). Coculture experiments showed that the number of viable THP-1 cells in the group containing adipocytes was more than that in the control group (P < 0.05). The difference between 3T3-L1 cell group and MSC group was statistically significant (P < 0.05).

CONCLUSIONThe ability of adipogenesis of 3T3-L1 cells is higher than that of human bone marrow MSCs in vitro. Adipocytes can protect THP-1 cell line against cytarabine, and the effect of adipocytes from 3T3-L1 cell group is greater than that from the human bone marrow MSC group.

3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Animals ; Bone Marrow Cells ; CCAAT-Enhancer-Binding Protein-alpha ; Cell Differentiation ; Fatty Acid-Binding Proteins ; Hematopoietic Stem Cells ; Humans ; Mesenchymal Stromal Cells ; Mice ; PPAR gamma ; RNA, Messenger

CCAAT/enhancer binding protein alpha gene (CEBPA) is an important transcription factor in maintenance of differentiation of granulocyte series of hematopoietic system. It plays a key role in regulating cell proliferation and differentiation. CEBPA mutation easily occurs in M1 and M2 type of acute myeloid leukemia, about 5%-14% in adult acute myeloid leukemia and 7.9% in children with acute myeloid leukemia. At present, domestic CEBPA mutation research is far less than abroad. This review focuses on the structual characteristics and detection method of CEBPA, CEBPA clinical features, the effect of CEBPA mutation on the prognosis of patients and the choice of treatment.
CCAAT-Enhancer-Binding Proteins ; Humans ; Leukemia, Myeloid, Acute ; Mutation ; Prognosis

OBJECTIVETo study the genetic pathogenesis of myasthenia gravis (MG) caused by cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene polymorphisms and regulation function of transcription factor.

METHODSELISA assay was used to determine the expression level of serum sCTLA-4 in MG. Four single nucleotide polymorphisms (SNPs) of CTLA-4 at exon 1 +49, promoter -318, -1661, -1772 were analyzed by restriction fragment length polymorphism (RFLP). Transcription factor nuclear factor 1(NF-1) and c/EBPbeta binding site were confirmed by chromatin immunoprecipitation(CHIP) assay.

RESULTSIt was found that the frequencies of the GG+49 genotype and G+49 allele are higher in MG patients with thymoma than those in patients of thymic hyperplasia and normal thymus subgroups. T/C-318 is not correlated with MG. The frequency of CT-1772 genotype is significantly higher in MG patients, especially in MG patients with thymoma, when compared with that in healthy controls. Meanwhile, the frequency of the G-1661 allele and GG-1661 genotype is lower in MG patients. Linkage disequilibrium (LD) between each SNPs in promoter -1772, -1661, -318 and coding sequence 1 (CDS 1) +49 is apparent. sCTLA-4 levels in patients' sera are correlated with the haplotype and genotype. T/C-1772 and A/G-1661 SNPs change the sequence of transcription factor NF-1 and c/EBPbeta binding sites. DNA variants lose site-specific binding activity of transcription factor regulated by lectin ConA and PHA.

CONCLUSIONThere are strong positive linkages among four SNPs. C/T-1772 and A/G-1661 polymorphisms can result in inefficient transcription of CTLA-4 gene. T>C-1772 mutation also affects gene splicing. These SNPs may constitute a factor of susceptibility to disease.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; Antigens, Differentiation ; blood ; genetics ; CCAAT-Enhancer-Binding Protein-beta ; genetics ; CCAAT-Enhancer-Binding Proteins ; genetics ; CTLA-4 Antigen ; Exons ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Myasthenia Gravis ; genetics ; immunology ; NFI Transcription Factors ; Point Mutation ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Thymoma ; genetics ; Thymus Hyperplasia ; genetics ; Thymus Neoplasms ; genetics ; Transcription Factors ; genetics

BACKGROUNDCCAAT/enhancer-binding protein beta (C/EBPbeta) is required for mitotic clonal expansion (MCE) during adipogenesis. It is still unclear how C/EBPbeta regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPbeta is required for preadipocyte proliferation, and identify new target genes of C/EBPbeta with chromatin immunoprecipitation (ChIP)-on-chip.

METHODSPostconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. ChIP was performed at 20 hours after induction with specific anti-C/EBPbeta antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.

RESULTSA total of 110 candidate genes were identified. BTG3 associated nuclear protein (SMAR1, Banp) and tripartite motif-containing 35 (Hls5, trim35) were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBPbeta to the promoter of banp and trim35 was verified by ChIP-PCR.

CONCLUSIONC/EBPbeta may regulate preadipocyte proliferation through activation of banp and trim35.

3T3-L1 Cells ; Adipocytes ; cytology ; Animals ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; CCAAT-Enhancer-Binding Protein-beta ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Differentiation ; genetics ; physiology ; Cell Proliferation ; Chromatin Immunoprecipitation ; DNA-Binding Proteins ; genetics ; metabolism ; Mice ; Nuclear Proteins ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; Protein Binding

BACKGROUND: The aim of this study was an in vitro investigation of the effect of high glucose concentration on adipogenesis, as prolonged hyperglycemia alters adipocyte differentiation. METHODS: 3T3-L1 preadipocytes differentiated in the presence of varying concentrations of glucose (25, 45, 65, 85, and 105 mM) were assessed for adipogenesis using AdipoRed (Lonza) assay. Cell viability and proliferation were measured using MTT reduction and [3H] thymidine incorporation assay. The extent of glucose uptake and glycogen synthesis were measured using radiolabelled 2-deoxy-D-[1-3H] glucose and [14C]-UDP-glucose. The gene level expression was evaluated using reverse transcription-polymerase chain reaction and protein expression was studied using Western blot analysis. RESULTS: Glucose at 105 mM concentration was observed to inhibit adipogenesis through inhibition of CCAAT-enhancer-binding proteins, sterol regulatory element-binding protein, peroxisome proliferator-activated receptor and adiponectin. High concentration of glucose induced stress by increasing levels of toll-like receptor 4, nuclear factor kappaB and tumor necrosis factor alpha thereby generating activated preadipocytes. These cells entered the state of hyperplasia through inhibition of p27 and proliferation was found to increase through activation of protein kinase B via phosphoinositide 3 kinase dependent pathway. This condition inhibited insulin signaling through decrease in insulin receptor beta. Although the glucose transporter 4 (GLUT4) protein remained unaltered with the glycogen synthesis inhibited, the cells were found to exhibit an increase in glucose uptake via GLUT1. CONCLUSION: Adipogenesis in the presence of 105 mM glucose leads to an uncontrolled proliferation of activated preadipocytes providing an insight towards understanding obesity.
Adipocytes ; Adipogenesis ; Adiponectin ; Blotting, Western ; CCAAT-Enhancer-Binding Proteins ; Cell Survival ; Glucose ; Glucose Transport Proteins, Facilitative ; Glycogen ; Hyperglycemia ; Hyperplasia ; Insulin ; Obesity ; Peroxisomes ; Phosphotransferases ; Proto-Oncogene Proteins c-akt ; Receptor, Insulin ; Thymidine ; Toll-Like Receptor 4 ; Tumor Necrosis Factor-alpha

CCAAT enhancer binding protein A (CEBPA) and its product transcription factor CCAAT enhancer binding protein &alpha; (C/EBP&alpha;) play pivotal roles in early granulocyte development. C/EBP&alpha; induces the transition and keeps the balance of differentiation and proliferation of myeloid progenitors. The mutation and dysregulation of CEBPA at transcription, translation or post-translation level lead to differentiation block and over proliferation of immature hematopoietic cells, which are important mechanisms of acute myeloid leukemia (AML). The mutation and dysregulation of CEBPA also provide clues for evaluating the outcome of AML patients and potential targets for differentiation-inducing therapies. This review focus on CEBPA mutation and AML, dysregulation of C/EBP&alpha; protein expression and AML, as well as C/EBP&alpha; protein and targeting therapy.
CCAAT-Enhancer-Binding Proteins ; genetics ; metabolism ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Mutation