Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 65
Filter
1.
Article in English | WPRIM | ID: wpr-879968

ABSTRACT

To investigate the effects of interleukin (IL)-17-mediated autophagy on the TNF receptor associated factor (TRAF6)/extracellular signal-regulated kinase (ERK)/p38 pathway and osteoclast differentiation. Mouse bone marrow-derived macrophages (BMM) were cultured with a medium containing 30 ng/mL macrophage colony stimulating factor and 50 ng/mL receptor activator of nuclear factor-kappa B ligard (RANKL), and IL-17 (0.01, 0.1, 1.0, 10 ng/mL) was added for intervention (IL-17 group). Tartrate-resistant acid phosphatase (TRAP) staining was used to observe TRAP positive multinucleated cells; phalloidin fluorescent staining was used to detect actin ring circumference; toluidine blue staining was used to analyze bone resorption lacuna formation. To further examine the mechanism of the effect of IL-17-mediated autophagy on the differentiation of osteoclasts, the control group used RANKL medium to culture mouse macrophage RAW264.7 cells, while the IL-17 group was treated with IL-17 (0.01, 0.1, 1.0, /mL). Western blot was used to detect the expression of autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) and osteoclast-related proteins c-fos and nuclear factor of activated T cell 1 (NFATc1) after treatment with different concentrations of IL-17. The expression of LC3, NFATc1, TRAF6/ERK/p38 signaling pathway related proteins were detected in IL-17 and autophagy inhibitor 3-MA group. The number of TRAP positive multinucleated cells, the circumference of the actin ring and the area of bone resorption lacuna in IL-17 group treated with IL-17 (0.01, 0.1, were significantly higher than those in the control group. In IL-17 treated RAW264.7 cells, the expression of c-fos, NFATc1, Beclin-1, LC3, TRAF6, p-ERK, and p-p38 was all significantly up-regulated (all 0.05). After treatment with the autophagy inhibitor 3-MA, the expression levels of LC3, NFATc1, TRAF6, p-ERK, and p-p38 all decreased significantly (all 0.05). IL-17 can promote the expression of autophagy proteins and enhance the differentiation ability of osteoclast precursor cells, and the TRAF6/ERK/p38 signaling pathway may be involved in this process.


Subject(s)
Animals , Autophagy , Bone Resorption , Cell Differentiation , Extracellular Signal-Regulated MAP Kinases , Interleukin-17 , Mice , NFATC Transcription Factors/metabolism , Osteoclasts/metabolism , RANK Ligand/metabolism , TNF Receptor-Associated Factor 6
2.
Article in Chinese | WPRIM | ID: wpr-880772

ABSTRACT

OBJECTIVE@#To investigate the effect of zoledronate (ZOL) on osteoclast differentiation and bone resorption under high glucose, and the regulation mechanism of p38 mitogen activated kinase (p38 MAPK) signaling pathway in this process.@*METHODS@#RAW264.7 cells were divided into four groups: low group, high group, low+ZOL group and high+ZOL group after induced into osteoclasts. Cell proliferation activity was determined by MTT assay. The migration of RAW264.7 cells were examined Optical microscopy. Immunofluorescence microscopy was used to observe the cytoskeleton and sealing zones of osteoclasts. After adding group 5: high + ZOL + SB203580 group, trap staining was used to identify the number of positive osteoclasts in each group. The number and area of resorption lacunae were observed by SEM. The mRNA and protein expression of osteoclast related factors were detected by real-time PCR and Western blotting.@*RESULTS@#The cells in the 5 groups showed similar proliferative activity. High glucose promoted the migration of RAW264.7 cells (@*CONCLUSIONS@#High glucose inhibits osteoclast differentiation and bone resorption. ZOL inhibits osteoclast differentiation and bone resorption in high-glucose conditions by regulating p38 MAPK pathway, which can be a new pathway for ZOL to regulate diabetic osteoporosis.


Subject(s)
Animals , Bone Resorption , Cell Differentiation , Glucose , MAP Kinase Signaling System , Mice , NFATC Transcription Factors , Osteoclasts , RANK Ligand , Zoledronic Acid/pharmacology , p38 Mitogen-Activated Protein Kinases
3.
Journal of Bone Metabolism ; : 113-121, 2019.
Article in English | WPRIM | ID: wpr-764243

ABSTRACT

BACKGROUND: Osteoporosis is a geriatric disease with diminished bone density. The increase in the number of patients and medical expenses due to a global aging society are recognized as problems. Bone loss is the most common symptom of bone disease, not only osteoporosis but Paget's disease, rheumatoid arthritis, multiple myeloma, and other diseases. The main cause of this symptoms is excessive increase in the number and activity of osteoclasts. Osteoclasts are multinucleated giant cells that can resorb bone. They are differentiated and activation from monocytes/macrophages in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). METHODS: The effect of extract of Flavoparmelia sp. (EFV), a genus of lichenized fungi within the Parmeliaceae, on the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts was examined by phenotype assay and the cell cytotoxicity was evaluated by cell counting kit-8. The osteoclast differentiation-related genes and proteins were investigated by real-time polymerase chain reaction and immunoblotting. The functional activity of osteoclast in response to EFV treatment was evaluated by an Osteo Assay plate. RESULTS: In this study, we found that EFV, a genus of lichenized fungi within the Parmeliaceae, inhibited osteoclast formation. And we investigated its inhibitory mechanism. EFV reduced RANKL-mediated osteoclast formation and activation by inhibiting expression of nuclear factor of activated T cells 1, a key factor of osteoclastogenesis. CONCLUSIONS: Taken together, our results show that EFV is a promising candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.


Subject(s)
Aging , Arthritis, Rheumatoid , Bone Density , Bone Diseases , Cell Count , Functional Food , Fungi , Giant Cells , Humans , Immunoblotting , Lichens , Macrophage Colony-Stimulating Factor , Macrophages , Multiple Myeloma , NFATC Transcription Factors , Osteoclasts , Osteoporosis , Parmeliaceae , Phenotype , Real-Time Polymerase Chain Reaction , T-Lymphocytes
4.
Article in Chinese | WPRIM | ID: wpr-771895

ABSTRACT

OBJECTIVE@#To explore the role of Ca-NFAT signaling pathway in Ph-ALL drug resistance mediated by bone marrow stromal cells.@*METHODS@#The transcription level of NFAT mRNA in Sup-B15 cells and Ph ALL primary cells was detected by polymerase chain reaction. The expression of P-glycoprotein in Sup-B15 cells was detected by flow cytometry. The change of NFAT protein in Sup-B15 cells was detected by Western blot. AnnexinV/7-AAD was used to label cells. Flow cytometry was used to detect cell apoptosis; Fluo 3-AM dye was used to label cells, and flow cytometry used to detect changes of Ca concentration in leukemia cells.@*RESULTS@#NFAT expression could be detected in both Sup-B15 and Ph ALL primary cells; P-glycoprotein could not be detected by flow cytometry; CAS could significantly inhibit NFAT protein expression in clinically applied drug concentrations (2.5, 5 μmol/L); Clinically applied concentration of CAS (2.5, 5 μmol / L) has no significant effect on the apoptosis of Sup-B15 cells, while higher concentration of CAS (10 μmol / L) could induce apoptosis of Sup-B15 cells. Bone marrow stromal cells OP9 could, decrease the sensitivity of Sup-B15 cells and Ph ALL primary cells to imatinib (IM); After co-culture with bone were marrow stromal cells, the Ca concentration in Sup-B15 cells was enhanced, the levels of NFAT protein and nullear protein in sup-B15 cells also were enhanced. The addition of CAS in co-culture system could inlibit the Ca-NFAT signaling pathway, reduce the protective effect of OP9 on Sup-B15 cells.Conclution:The Ca-NFAT sigualing pathway, contributes to the survival of Ph ALL cells. Bone marrow stromal cells can mediate the resistance of Ph ALL cells to IM by activating Ca-NFAT signaling pathway.


Subject(s)
Bone Marrow Cells , Cell Line, Tumor , Humans , Imatinib Mesylate , Mesenchymal Stem Cells , NFATC Transcription Factors , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Signal Transduction
5.
Article in Chinese | WPRIM | ID: wpr-773550

ABSTRACT

OBJECTIVE@#To explore the mechanism by which simvastatin (SIM) regulates osteoclast apoptosis.@*METHODS@#Murine macrophage RAW264.7 cells were divided into 5 groups, namely group A (control group), group B (sRANKL+ M-CSF), group C (SIM+sRANKL+M-CSF), group D (VIVIT peptide+sRANKL+ M-CSF), and group E (SIM+VIVIT peptide+sRANKL+M-CSF). WST-1 assay was used to assess the effects of simvastatin on the proliferation activity of the osteoclasts, and flow cytometry was performed to analyze the effects of SIM and VIVIVIT peptide (a NFATc1 pathway inhibitor) on apoptosis of the osteoclasts. The translocation of NFATc1 into the nucleus was investigated using immunofluorescence assay, and Western blotting was employed to assess the effect of SIM on the phosphorylation of NFATc1 in the nucleus.@*RESULTS@#WST-1 assay showed that SIM (1×10 mol/L) treatment for 24 and 48 h significantly inhibited the proliferation of the osteoclasts (=0.039 and 0.022, respectively). Compared with the control group, the SIM-treated osteoclasts exhibited significantly reduced cell percentage in G0/G1 phase (=0.041) and increased cells in sub-G1 phase (=0.028) with obvious cell apoptosis. DAPI staining and flow cytometry showed that both SIM and VIVIVIT peptide alone significantly promoted osteoclast apoptosis (=0.002 and 0.015, respectively), and their combination produced a similar pro-apoptosis effect (=0.08). Immunofluorescence and Western blotting showed that SIM significantly inhibited the intranuclear translocation of NFATc1 and the phosphorylation of NFATc1 pathway protein (=0.013).@*CONCLUSIONS@#SIM promotes osteoclast apoptosis through NFATc1 signaling pathway.


Subject(s)
Animals , Apoptosis , Cell Differentiation , Mice , NFATC Transcription Factors , Osteoclasts , RANK Ligand , Simvastatin
6.
Biol. Res ; 52: 10, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011412

ABSTRACT

BACKGROUND: Non-canonical Wnt pathways play important roles in liver fibrosis. Notum is a newly discovered inhibitor to Wnt proteins. This study was to investigate anti-fibrotic effects of Notum. METHODS: 53 patients with hepatitis B virus (HBV) infection as well as a cell co-culture system of LX-2 and Hep AD38 cells were engaged in this study. Clinical, biological and virological data of each patient were analyzed. Cell viability was detected at different time points. mRNA and protein levels of NFATc1 (Nuclear factor of activated T-cells), Jnk, α-SMA, Col1A1 and TIMP-1 were detected both in LX-2 and liver tissue. Protein levels of NFATc1 and Jnk in liver tissue and their correlations with fibrosis score were analyzed. RESULTS: Hepatitis B virus replication up-regulated Wnt5a induced NFATc1 and Jnk activity in Hep AD38. Notum suppressed NFATc1, Jnk and fibrosis genes expression, reduced cell viability in co-cultured LX-2 cells induced by HBV. Interestingly, Patients with HBV DNA > 5log copies/ml had higher mRNA levels of NFATc1 and fibrosis genes than patients with HBV DNA < 5log copies/ml. Most importantly, protein expressions of NFATc1 and pJnk have positive correlations with liver fibrosis scores in HBV-infected patients. CONCLUSIONS: Our data showed that Notum inhibited HBV-induced liver fibrosis through down-regulating Wnt 5a mediated non-canonical pathways. This study shed light on anti-fibrotic treatment.


Subject(s)
Humans , Male , Female , Adult , Esterases/administration & dosage , Wnt-5a Protein/antagonists & inhibitors , Hepatitis B/complications , Liver Cirrhosis/prevention & control , Virus Replication , Transfection , Cell Survival , Hepatitis B virus/physiology , Actins/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism , Collagen Type I/metabolism , MAP Kinase Kinase 4/metabolism , NFATC Transcription Factors/analysis , NFATC Transcription Factors/metabolism , Wnt Signaling Pathway , Wnt-5a Protein/metabolism , Liver Cirrhosis/metabolism , Liver Cirrhosis/virology
7.
Article in Chinese | WPRIM | ID: wpr-771479

ABSTRACT

OBJECTIVE@#To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes.@*METHODS@#The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting.@*RESULTS@#Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05).@*CONCLUSIONS@#Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.


Subject(s)
Aldehyde Dehydrogenase, Mitochondrial , Metabolism , Animals , Animals, Newborn , Benzamides , Pharmacology , Benzodioxoles , Pharmacology , Calcium , Metabolism , Cells, Cultured , Culture Media , Enzyme Inhibitors , Pharmacology , Glucose , Pharmacology , Isoflavones , Pharmacology , Mitochondria, Heart , Myocytes, Cardiac , Metabolism , NFATC Transcription Factors , Metabolism , Nuclear Pore Complex Proteins , Metabolism , Rats , Rats, Sprague-Dawley
8.
Article in Chinese | WPRIM | ID: wpr-773808

ABSTRACT

OBJECTIVE@#To investigate the effects of hydrogen sulfide (HS) on the negatively regulation of cardiomyocyte hypertrophy and the relationship between the effect of HS with miRNA-133a-mediated Ca/calcineurin/NFATc4 signal pathway.@*METHODS@#Cardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (Leica). The expression of brain natriuretic peptide(BNP), β-myosin heavy chain(β-MHC), cystathionase (CSE), miRNA-133a, calcineurin (CaN) were detected by qRT-PCR. The protein expressions of CaN、nuclear factors of activated T cells (NFATc4) were detected by Western blot. The concentration of HS in the cardiomyocyte was detected by Elisa. The concentration of intracellular calcium was measured by calcium imaging using confocal microscope. The nuclear translocation of NFATc4 was checked by immuno-fluorescence cell staining technique.@*RESULTS@#①The level of system of CSE/HS and expression of miRNA-133a were significantly reduced in cardiomyocyte hypertrophy. Pretreatment with NaHS increased the concentration of HS and the expression of miRNA-133a mRNA in cardiomyocytes, and suppressed cardiomyocyte hypertrophy. ②The concentration of intracellular calcium, the expression of CaN and nulear protein NFATc4 were significantly increased, and the nuclear translocation of NFATc4 were obviously enhanced in cardiomyocyte hypertrophy. NaHS pretreatment markedly inhibited these effects of ISO induced cardiomyocyte hypertrophy. ③Application of antagomir-133a reversed the inhibitory effects of NaHS on cardiomyocyte hypertrophy, and increased the influx of intracellular calcium, and elevated the expression of CaN and nuclear protein NFATc4, and enhanced the nuclear translocation of NFATc4.@*CONCLUSIONS@#HS can negatively regulate cardiomyocyte hypertrophy. The effects might be associated with HS increasing expression of miRNA-133a and inhibiting inactivation of Ca/calcineurin/NFATc4 signal pathway.


Subject(s)
Animals , Calcineurin , Metabolism , Cardiomegaly , Metabolism , Cells, Cultured , Cystathionine gamma-Lyase , Metabolism , Hydrogen Sulfide , Metabolism , MicroRNAs , Metabolism , Myocytes, Cardiac , Metabolism , Myosin Heavy Chains , Metabolism , NFATC Transcription Factors , Metabolism , Natriuretic Peptide, Brain , Metabolism , Nerve Tissue Proteins , Metabolism , Rats , Signal Transduction
9.
Mem. Inst. Oswaldo Cruz ; 113(9): e180171, 2018. graf
Article in English | LILACS | ID: biblio-955120

ABSTRACT

BACKGROUND The anti-inflammatory and cardioprotective properties of curcumin (Cur), a natural polyphenolic flavonoid isolated from the rhizomes of Curcuma longa, are increasingly considered to have beneficial effects on the progression of Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi. OBJECTIVE To evaluate the effects of oral therapy with Cur on T. cruzi-mediated cardiovasculopathy in acutely infected mice and analyse the in vitro response of parasite-infected human microvascular endothelial cells treated with this phytochemical. METHODS Inflammation of heart vessels from Cur-treated and untreated infected mice were analysed by histology, with benznidazole (Bz) as the reference compound. Parasitaemia was monitored by the direct method. Capillary permeability was visualised by Evans-blue assay. Myocardial ET-1, IL-6, and TNF-α mRNA expressions were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Microvascular endothelial HMEC-1 cells were infected in vitro with or without addition of Cur or Bz. Induction of the Ca2+/NFAT pathway was assessed by fluorometry, immunoblotting, and reporter assay. FINDINGS Oral Cur therapy of recently infected mice reduced inflammatory cell infiltration of myocardial arteries without lowering parasite levels. Compared to that of the phosphate-buffered saline-receiving group, hearts from Cur-treated mice showed significantly decreased vessel inflammation scores (p < 0.001), vascular permeabilities (p < 0.001), and levels of IL-6/TNF-α (p < 0.01) and ET-1 (p < 0.05) mRNA. Moreover, Cur significantly (p < 0.05 for transcript; p < 0.01 for peptide) downregulated ET-1 secretion from infected HMEC-1 cells. Remarkably, Cur addition significantly (p < 0.05 at 27.0 μM) interfered with T. cruzi-dependent activation of the Ca2+/NFATc1 signalling pathway that promotes generation of inflammatory agents in HMEC-1 cells. CONCLUSIONS Oral treatment with Cur dampens cardiovasculopathy in acute Chagas mice. Cur impairs the Ca2+/NFATc1-regulated release of ET-1 from T. cruzi-infected vascular endothelium. These findings identify new perspectives for exploring the potential of Cur-based interventions to ameliorate Chagas heart disease.


Subject(s)
Humans , Chagas Cardiomyopathy/parasitology , Reverse Transcriptase Polymerase Chain Reaction , NFATC Transcription Factors/analysis , Blotting, Western , Acute Disease/rehabilitation , Anti-Inflammatory Agents/pharmacology
10.
Article in English | WPRIM | ID: wpr-812100

ABSTRACT

Inflammation is recently recognized as one of the hallmarks of human cancer. Chronic inflammatory response plays a critical role in cancer development, progression, metastasis, and resistance to chemotherapy. Conversely, the oncogenic aberrations also generate an inflammatory microenvironment, enabling the development and progression of cancer. The molecular mechanisms of action that are responsible for inflammatory cancer and cancer-associated inflammation are not fully understood due to the complex crosstalk between oncogenic and pro-inflammatory genes. However, molecular mediators that regulate both inflammation and cancer, such as NF-κB and STAT have been considered as promising targets for preventing and treating these diseases. Recent works have further demonstrated an important role of oncogenes (e.g., NFAT1, MDM2) and tumor suppressor genes (e.g., p53) in cancer-related inflammation. Natural products that target these molecular mediators have shown anticancer and anti-inflammatory activities in preclinical and clinical studies. Sesquiterpenoids (STs), a class of novel plant-derived secondary metabolites have attracted great interest in recent years because of their diversity in chemical structures and pharmacological activities. At present, we and other investigators have found that dimeric sesquiterpenoids (DSTs) may exert enhanced activity and binding affinity to molecular targets due to the increased number of alkylating centers and improved conformational flexibility and lipophilicity. Here, we focus our discussion on the activities and mechanisms of action of STs and DSTs in treating inflammation and cancer as well as their structure-activity relationships.


Subject(s)
Animals , Anti-Inflammatory Agents , Pharmacology , Antineoplastic Agents, Phytogenic , Pharmacology , Humans , Inflammation , Drug Therapy , NF-kappa B , NFATC Transcription Factors , Neoplasms , Drug Therapy , Proto-Oncogene Proteins c-mdm2 , Physiology , Sesquiterpenes , Chemistry , Pharmacology , Structure-Activity Relationship
11.
Rio de Janeiro; s.n; 2017. ilus, tab, graf.
Thesis in Portuguese | LILACS, Inca | ID: biblio-943739

ABSTRACT

A família de fatores de transcrição NFAT (Fator Nuclear de células T Ativadas) é composta por quatro proteínas responsivas a influxo de cálcio (NFAT1-4) que apresentam diferentes isoformas já descritas. O NFAT2 foi correlacionado com alguns tipos de cânceres como linfomas e carcinomas pancreáticos, sugerindo um papel desta proteína na oncogênese. Duas isoformas do NFAT2 divergem apenas em sua porção N-terminal, apresentando 42 aminoácidos diferenciais no N-terminal da isoforma NFAT2a, codificados pelo éxon 1 do gene e 28 aminoácidos diferenciais no NFAT2b, codificados pelo éxon 2. Em linfócitos T e B naïve, o NFAT2b apresenta níveis basais de expressão,enquanto os níveis de NFAT2a são massivamente aumentados após ativação antigênica. Apesar destes perfis distintos de expressão sugerirem funções diferenciais na regulação gênica, pouco se conhece a respeito de papéis específicos destas isoformas na fisiologia celular. O objetivo destetrabalho foi caracterizar o envolvimento das isoformas NFAT2a e NFAT2b no controle de morte e transformação celular. Para investigar os papéis diferenciais das isoformas do NFAT2, formas constitutivamente ativas (CA) destas proteínas foram expressas em fibroblastos NIH3T3 por transdução retroviral. Nossos dados mostraram que as proteínas CA-NFAT2a e CA-NFAT2b afetam diferencialmente a transformação e a morte celular em células NIH3T3. Células expressando CANFAT2 a apresentaram fenótipos de transformação celular como perda da inibição de crescimento por contato, crescimento independente de ancoragem e formação de tumores em camundongos imunodeficientes após inoculação subcutânea. Em contrapartida, CA-NFAT2b levou a um fenótipo menos intenso de transformação celular e induziu morte celular a partir da regulação positiva dacitocina TNF-a...


NFAT (Nuclear Factor of Activated T cells) family of transcription factors is composed of four calcium responsive proteins (NFAT1-4) that display different isoforms. NFAT2 were associated to the development of several cancer types such as lymphomas and pancreatic carcinomas. Two of the NFAT2 isoforms diverge only at the N terminus, which contains 42 amino acids in the NFAT2a protein, encoded by exon 1 of the gene, or 28 differential amino acids in the NFAT2b protein, encoded by exon 2. In naïve T and B lymphocytes, NFAT2b has a basal expression level while NFAT2a levels are massively increased after antigenic activation. Although distinct expression patterns suggest differential roles in gene regulation, specific roles in cell physiology for NFAT2 isoforms remainunclear. Then, the aim of this work is to analyze differential roles of NFAT2 isoforms in cell death and transformation. In order to study the roles of NFAT2 isoforms in cell physiology, non-transformed NIH3T3 fibroblasts were retrovirally transduced with vectors containing constitutively active (CA) formsof NFAT2 proteins. Our data showed that CA-NFAT2a and CA-NFAT2b distinctly affect cell death and transformation in NIH3T3 cells. CA-NFAT2a-expressing cells showed cell transformation phenotypes such as loss of contact growth inhibition, anchorage-independent cell growth, and tumor formation in immunodeficient mice. In contrast, CA-NFAT2b led to a milder cell-transformation phenotype andinduced cell death through up-regulation of TNF-a cytokine. Furthermore, we demonstrated that differential roles for NFAT2 isoforms in NIH3T3 cells depend of alternative N-terminal domains. A NFAT2b-specific N-terminal acidic domain is necessary for cell death induction, whereas its ablation or substitutions of conserved acidic amino acids completely abolish cell death and enhance cell transformation phenotype. CA-NFAT2b expression also induces cell death and increases the levels of FasL and TNF-a in CD4+ T cells...


Subject(s)
Humans , Male , Female , Mice , Cell Death , Gene Expression , NFATC Transcription Factors , Protein Isoforms
12.
Article in Chinese | WPRIM | ID: wpr-232519

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the role of nuclear factor of activated T cells -2 (NFAT2) in release of high mobility protein box-1 (HMGB1) from human monocytic THP-1 cells in vitro.</p><p><b>METHODS</b>The level of HMGB1 release from THP-1 cells in response to lipopolysaccharide (LPS) stimulation was examined by Western blotting and enzyme-linked immunosorbent assay (ELISA). The effect of LPS stimulation on NFAT2 and HMGB1 interaction in the cytoplasm was observed by immunoprecipitation assay. HMGB1 production and release was detected in cells with specific small interfering RNA (siRNA)-mediated suppression of NFAT2 expression.</p><p><b>RESULTS</b>LPS stimulated HMGB1 release from THP-1 cells. As LPS stimulation prolonged, HMGB1 concentration increased in the cell culture supernatant and decreased in the cytoplasm, and the binding between NFAT2 and HMGB1 was not detected in the cell nuclei. NFAT2 suppression by the siRNA plasmid resulted in increased HMGB1 level in the cell culture supernatant.</p><p><b>CONCLUSION</b>NFAT2 can inhibit HMGB1 release from THP-1 cells in vitro.</p>


Subject(s)
Blotting, Western , Cell Line , Cell Nucleus , Cytoplasm , Metabolism , Enzyme-Linked Immunosorbent Assay , HMGB1 Protein , Metabolism , Humans , Lipopolysaccharides , Monocytes , Metabolism , NFATC Transcription Factors , Metabolism
13.
J. appl. oral sci ; 23(6): 549-554, Nov.-Dec. 2015. graf
Article in English | LILACS, BBO | ID: lil-769817

ABSTRACT

ABSTRACT Objective This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). Material and Methods The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. Results After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. Conclusions Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.


Subject(s)
Humans , /metabolism , Osteogenesis/physiology , Osteoprotegerin/metabolism , Periodontal Ligament/cytology , Benzylamines/pharmacokinetics , Blotting, Western , Bone Resorption/metabolism , Cells, Cultured , Down-Regulation , NFATC Transcription Factors/metabolism , Pressure , Protein Kinase Inhibitors/pharmacokinetics , RANK Ligand/analysis , RANK Ligand/metabolism , Random Allocation , Real-Time Polymerase Chain Reaction , Sulfonamides/pharmacokinetics , Time Factors , Up-Regulation
14.
Dental press j. orthod. (Impr.) ; 20(1): 79-84, Jan-Feb/2015. tab, graf
Article in English | LILACS | ID: lil-741451

ABSTRACT

OBJECTIVE: The aim of the present study was to determine the morphological differences in the base of the skull of individuals with cleft lip and palate and Class III malocclusion in comparison to control groups with Class I and Class III malocclusion. METHODS: A total of 89 individuals (males and females) aged between 5 and 27 years old (Class I, n = 32; Class III, n = 29; and Class III individuals with unilateral cleft lip and palate, n = 28) attending PUC-MG Dental Center and Cleft Lip/Palate Care Center of Baleia Hospital and PUC-MG (CENTRARE) were selected. Linear and angular measurements of the base of the skull, maxilla and mandible were performed and assessed by a single calibrated examiner by means of cephalometric radiographs. Statistical analysis involved ANCOVA and Bonferroni correction. RESULTS: No significant differences with regard to the base of the skull were found between the control group (Class I) and individuals with cleft lip and palate (P > 0.017). The cleft lip/palate group differed from the Class III group only with regard to CI.Sp.Ba (P = 0.015). Individuals with cleft lip and palate had a significantly shorter maxillary length (Co-A) in comparison to the control group (P < 0.001). No significant differences were found in the mandible (Co-Gn) of the control group and individuals with cleft lip and palate (P = 1.000). CONCLUSION: The present findings suggest that there are no significant differences in the base of the skull of individuals Class I or Class III and individuals with cleft lip and palate and Class III malocclusion. .


OBJETIVO: o objetivo do presente estudo foi determinar diferenças morfológicas da base do crânio de indivíduos portadores de fissura de lábio e palato e de má oclusão de Classe III, comparado-os com indivíduos controle com má oclusão de Classes I ou III. MÉTODOS: oitenta e nove indivíduos, de ambos os sexos, com idade variando entre 5 e 27 anos, Classe I (n = 32), Classe III não fissurados (n = 29) e Classe III com fissura labiopalatina unilateral (n = 28), oriundos do Centro de Odontologia e Pesquisa da PUC-MG e do Centro de Atendimento de Fissurados do Hospital da Baleia e da PUC-MG (CENTRARE), foram selecionados. Medições lineares e angulares da base do crânio, maxila e mandíbula foram realizadas e avaliadas por um único examinador calibrado, por meio de radiografias cefalométricas. Foram utilizados os testes ANCOVA e correção de Bonferroni para a análise estatística dos dados. RESULTADOS: com relação à base do crânio, os resultados não indicaram diferença estatística entre indivíduos controle (Classe I) e os indivíduos com fissuras (p > 0,017). O grupo com fissura foi diferente do grupo Classe III somente em relação à medida CI.Sp.Ba (p = 0,015). O comprimento maxilar (Co-A) apresentou diferença estatisticamente significativa na comparação entre o grupo controle (Classe I) e o grupo com fissuras (p < 0,001), sendo que os fissurados apresentaram uma maxila menor. Não foram encontradas diferenças na mandíbula (Co-Gn) entre indivíduos do grupo controle (Classe I) e indivíduos fissurados (p = 1,000). CONCLUSÃO: os resultados sugerem que não houve diferença estatisticamente significativa na base do crânio entre indivíduos Classe I e III e indivíduos com fissuras de lábio e palato com má oclusão de Classe III. .


Subject(s)
Animals , Female , Cardiomegaly/metabolism , Cardiomegaly/pathology , Fetal Heart/metabolism , Fetal Heart/pathology , Maternal Nutritional Physiological Phenomena , Overnutrition/metabolism , Overnutrition/pathology , Biomarkers/metabolism , Calcineurin/metabolism , Cardiovascular Diseases/epidemiology , Extracellular Space , Fascia/pathology , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental , Myofibrils/pathology , NFATC Transcription Factors/metabolism , Natriuretic Peptides/genetics , Natriuretic Peptides/metabolism , Phosphorylation , RNA, Messenger/metabolism , Sheep, Domestic , TOR Serine-Threonine Kinases/metabolism
15.
Article in English | WPRIM | ID: wpr-150122

ABSTRACT

Osteoclasts are multinucleated cells formed mainly on bone surfaces in response to cytokines by fusion of bone marrow-derived myeloid lineage precursors that circulate in the blood. Major advances in understanding of the molecular mechanisms regulating osteoclast formation and functions have been made in the past 20 years since the discovery that their formation requires nuclear factor-kappa B (NF-kappaB) signaling and that this is activated in response to the essential osteoclastogenic cytokine, receptor activator of NF-kappaB ligand (RANKL), which also controls osteoclast activation to resorb (degrade) bone. These studies have revealed that RANKL and some pro-inflammatory cytokines, including tumor necrosis factor, activate NF-kappaB and downstream signaling, including c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and inhibition of repressors of NFATc1 signaling, to positively regulate osteoclast formation and functions. However, these cytokines also activate NF-kappaB signaling that can limit osteoclast formation through the NF-kappaB signaling proteins, TRAF3 and p100, and the suppressors of c-Fos/NFATc1 signaling, IRF8, and RBP-J. This paper reviews current understanding of how NF-kappaB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast formation and activation.


Subject(s)
Cytokines , NF-kappa B , NFATC Transcription Factors , Osteoclasts , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , TNF Receptor-Associated Factor 3 , Tumor Necrosis Factor-alpha
16.
Article in English | WPRIM | ID: wpr-119559

ABSTRACT

Osteoclasts originated from hematopoietic stem cells are multi-nucleated cells that can resorb the bone matrix. Receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) signaling pathway is crucial for the differentiation and activation of osteoclasts. In this study, we investigated for the first time whether or not RANKL induced mitogen- and stress-activated kinase 1 (MSK1) phosphorylation at Ser 376. Activation of MSK1 was detected as soon as 5 min after RANKL stimulation and sparsely detected at 30 min after stimulation. RANKL-induced MSK1 phosphorylation occurred in a dose-dependent manner. MSK1 is known as a downstream signaling molecule of cAMP-dependent protein kinase (PKA). Treatment with the PKA inhibitor H89 significantly suppressed c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) induction upon RANKL stimulation. In addition, cAMP response element-binding protein (CREB) phosphorylation was extremely inhibited by H89 treatment. Mitogen-activated protein kinases (MAPKs) have been investigated for induction of MSK1 phosphorylation. Specific signaling pathway inhibitors for p38 and extracellular signal-regulated kinases (ERKs) significantly blocked RANKL-induced MSK1 activation. Finally, as a downstream effector of the p38-MSK1 pathway, c-Fos transcriptional activity was determined. RANKL-mediated elevation of c-Fos transcriptional activity was significantly suppressed by p38 inhibitor. Moreover, a dominant negative form of CREB suppressed activation of NFATc1. In conclusion, RANKL-stimulated MSK1 phosphorylation could play a role in induction of NFATc1 through CREB and c-Fos activation as a downstream molecule of p38, ERK MAPKs, and PKA. Our results support basic information for the development of osteoclast specific inhibitors.


Subject(s)
Bone Matrix , Cyclic AMP Response Element-Binding Protein , Cyclic AMP-Dependent Protein Kinases , Extracellular Signal-Regulated MAP Kinases , Hematopoietic Stem Cells , Mitogen-Activated Protein Kinases , NFATC Transcription Factors , Osteoclasts , Phosphorylation , Phosphotransferases
17.
Article in Chinese | WPRIM | ID: wpr-346170

ABSTRACT

<p><b>OBJECTIVE</b>To study the expression of Foxp3 and NFAT1 protein in peripheral blood (PB) in children with aplastic anemia (AA) and their roles in the pathogenesis of AA.</p><p><b>METHODS</b>The expression levels of Foxp3 and NFAT1 protein of mononuclear cells in PB were measured by Western blot in 68 children with AA before and after treatment and in 60 normal children (control group). The correlation between Foxp3 and NFAT1 protein expression and the correlation of the Foxp3 and NFAT1 protein expression with blood Hb, WBC and platelet levels were analyzed.</p><p><b>RESULTS</b>The expression levels of Foxp3 and NFAT1 protein in PB in the acute phase in the AA group were significantly lower than in the control group (P<0.05). After treatment (recovery phase) the expression levels of Foxp3 and NFAT1 protein increased obviously compared with those in the acute phase (P<0.05). The Foxp3 protein level was positively correlated with the NFAT1 protein level (r=0.812, P<0.05). Both the Foxp3 and NFAT1 protein levels were positively correlated with blood Hb, WBC and platelet levels in children with AA in the recovery phase (r=0.537, 0.579, 0.655 respectively; P<0.05).</p><p><b>CONCLUSIONS</b>The Foxp3 and NFAT1 protein levels in PB are reduced in children with AA, suggesting that they are involved in the pathogenesis of AA. The measurement of Foxp3 and NFAT1 protein levels may be useful in the severity evaluation of AA.</p>


Subject(s)
Adolescent , Anemia, Aplastic , Blood , Child , Child, Preschool , Female , Forkhead Transcription Factors , Blood , Humans , Male , NFATC Transcription Factors , Blood
18.
Article in Chinese | WPRIM | ID: wpr-317811

ABSTRACT

<p><b>OBJECTIVE</b>This study investigated the role and mechanism of calcineurin (CaN)-nuclear factor of activated T cells (NFAT) pathway in the myoblast apoptosis induced by cyclic tensile strain.</p><p><b>METHODS</b>Myoblasts were cultured using an in vitro-mechanical stimulation model and imposed with tension for different hours with a multi-channel cell stress loading system. Cyclosporine (CsA) was used as CaN inhibitor to clarify the role of CaN in the apoptosis induced by cyclic stress. Hochest 33258 staining and flow cytometry detection were performed to detect the apoptotic cells. Real-time polymerase chain reaction was conducted to detect the mRNA expression of CaN and NFAT. Protein levels of NFAT3 were evaluated by Western blot.</p><p><b>RESULTS</b>The apoptosis rate increased with the extension of loading time. The mRNA expression of the CaN subunits, CnA and CnB, and the protein levels of NFAT3 also increased. When the myoblasts were incubated with CsA, the apoptosis rate decreased, the mRNA expression of CnA and NFAT3 significantly decreased, and the NFAT3 protein expression levels became significantly lower than those of the groups without CsA.</p><p><b>CONCLUSION</b>Continuous cyclic tensile stress can induce myoblast apoptosis. The CaN-NFAT signaling pathway may be involved in the cyclic stretch-induced apoptosis of myoblasts.</p>


Subject(s)
Apoptosis , Calcineurin , Genetics , Cyclosporine , Flow Cytometry , Myoblasts , Physiology , NFATC Transcription Factors , Metabolism , Real-Time Polymerase Chain Reaction , Signal Transduction , T-Lymphocytes
19.
Chinese Journal of Cardiology ; (12): 614-618, 2015.
Article in Chinese | WPRIM | ID: wpr-317706

ABSTRACT

<p><b>OBJECTIVE</b>To observe whether CD137 signaling could affect the nuclear factor of activated T cells c1 (NFATc1) expression through nuclear factor-κB (NF-κB) pathway in mice aortic vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>Adherence methods for tissues explants were used for primary culture of mouse aortic VSMCs. The mRNA expression of CD137 and NFATc1 was detected by real-time quantitative PCR (RT-qPCR). The VSMCs protein expression of IκB-α, NF-κB p65, phospo-p65 and NFATc1 was determined by Western blot. The level of CD137 was measured by Flow Cytometry (FCM).</p><p><b>RESULTS</b>(1) The mRNA and protein expression of CD137 in VSMCs was significantly upregulated at 24 h after co-culture with TNF-α (10 ng/ml, all P < 0.05). (2) Compared with the control group, the level of p-NF-κB p65 in cytoplasm and nucleus was significantly increased (8.34 ± 0.28 vs. 1, P < 0.05, and 2.64 ± 0.42 vs. 1, P < 0.05) while the level of IκB-α was reduced (1 vs. 2.70 ± 0.28, P < 0.05) after co-treatment with agonist-CD137 mAb, above effects were partly blocked by adding specific NF-κB inhibitor PDTC (30 µmol/L: 1.15 ± 0.14 vs. 8.34 ± 0.28, P < 0.05, and 2.09 ± 0.12 vs. 2.64 ± 0.42, P < 0.05, and 1.78 ± 0.74 vs. 1, P < 0.05). (3) The mRNA (2.07 ± 0.09 vs. 1, P < 0.05) and protein (1.75 ± 0.07 vs. 1, P < 0.05) expression of NFATc1 was significantly upregulated by agonist CD137mAb compared with the control group, and these effects could be reversed by PDTC (1.15 ± 0.07 vs. 2.07 ± 0.09, P < 0.05, and 0.90 ± 0.11 vs. 1.75 ± 0.07, P < 0.05).</p><p><b>CONCLUSION</b>CD137 signaling could affect the NFATc1expression in VSMCs through NF-kappaB pathway.</p>


Subject(s)
Animals , Cells, Cultured , I-kappa B Proteins , Mice , Muscle, Smooth, Vascular , Metabolism , Myocytes, Smooth Muscle , NF-KappaB Inhibitor alpha , NF-kappa B , Metabolism , NFATC Transcription Factors , Metabolism , RNA, Messenger , Signal Transduction , Tumor Necrosis Factor Receptor Superfamily, Member 9 , Metabolism , Tumor Necrosis Factor-alpha , Up-Regulation
20.
Chinese Journal of Cardiology ; (12): 887-893, 2015.
Article in Chinese | WPRIM | ID: wpr-317669

ABSTRACT

<p><b>OBJECTIVE</b>To investigate if miR-145a-5p participates the modulation process of CD137 signaling on the expression of nuclear factor of activated T cells c1 (NFATc1) in ApoE(-)/(-) mice.</p><p><b>METHODS</b>Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE(-)/(-) mice. After surgery, the mice were randomly divided into the following groups: CD137 activated group (CD137 group, n = 6), CD137 inhibited group (anti-CD137 group, n = 6) and control group (n = 6). The mRNA expression of miR-145a-5p in plaque and cells was measured by real-time quantitative PCR (RT-PCR). Immunofluorescence was used to observe the distribution of NFATc1 in plaque and the expression of NFATc1 at mRNA and protein levels were detected by qRT-PCR, Western blot, respectively. The mouse vascular smooth muscle cells (VSMCs) were isolated and transfected with miR-145a-5p mimics or inhibitors by Lipofectamine. The eukaryotic expression vector and luciferase vector including p3xFLAG-NFATc1, p3xFLAG-NFATc1-3'UTR, psicheck2-NFATc1, psicheck2-NFATc1-Mut were constructed through molecular cloning and homologous recombination techniques, 293T cells were transfected with the miR-145a-5p mimics or inhibitors and the protein level and fluorescence intensity were then measured, respectively.</p><p><b>RESULTS</b>In vivo or in vitro, the level of miR-145a-5p was significantly decreased (0.21 ± 0.06 vs. 1.00 ± 0.00, P < 0.05, 0.22 ± 0.07 vs. 0.50 ± 0.12, P < 0.05) while the opposite effects were observed in anti-CD137 group. NFATc1 expression was decreased or increased in VSMCs transfected with miR-145a-5p mimics or inhibitors, respectively (all P < 0.05). miR-145a-5p mimics decreased the expression of p3xFLAG-NFATc1-3'UTR and the fluorescence intensity (0.56 ± 0.08 vs. 1.00 ± 0.00, P < 0.05).</p><p><b>CONCLUSION</b>CD137 signaling participates the regulation process on the expression of NFATc1 through miR-145a-5p in ApoE(-)/(-) mice.</p>


Subject(s)
Animals , Apolipoproteins E , Mice , Mice, Knockout , MicroRNAs , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , NFATC Transcription Factors , Plaque, Atherosclerotic , RNA, Messenger , Signal Transduction , T-Lymphocytes , Transfection , Tumor Necrosis Factor Receptor Superfamily, Member 9
SELECTION OF CITATIONS
SEARCH DETAIL