scholarly journals Vasopressin-dependent Myogenic Cell Differentiation Is Mediated by Both Ca2+/Calmodulin-dependent Kinase and Calcineurin Pathways

2005 ◽  
Vol 16 (8) ◽  
pp. 3632-3641 ◽  
Author(s):  
Bianca Maria Scicchitano ◽  
Lucia Spath ◽  
Antonio Musarò ◽  
Mario Molinaro ◽  
Nadia Rosenthal ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Myogenic Cell ◽  
Specific Gene ◽  
Myocyte Enhancer Factor 2 ◽  
First Time ◽  
Kinetics Of ◽  
Enhancer Factor ◽  
Formation Of Complexes

Arg8-vasopressin (AVP) promotes the differentiation of myogenic cell lines and mouse primary satellite cells by mechanisms involving the transcriptional activation of myogenic bHLH regulatory factors and myocyte enhancer factor 2 (MEF2). We here report that AVP treatment of L6 cells results in the activation of calcineurin-dependent differentiation, increased expression of MEF2 and GATA2, and nuclear translocation of the calcineurin target NFATc1. Interaction of these three factors occurs at MEF2 sites of muscle specific genes. The different kinetics of AVP-dependent expression of early (myogenin) and late (MCK) muscle-specific genes correlate with different acetylation levels of histones at their MEF2 sites. The cooperative role of calcineurin and Ca2+/calmodulin-dependent kinase (CaMK) in AVP-dependent differentiation is demonstrated by the effect of inhibitors of the two pathways. We show here, for the first time, that AVP, a “novel” myogenesis promoting factor, activates both the calcineurin and the CaMK pathways, whose combined activation leads to the formation of multifactor complexes and is required for the full expression of the differentiated phenotype. Although MEF2–NFATc1 complexes appear to regulate the expression of an early muscle-specific gene product (myogenin), the activation of late muscle-specific gene expression (MCK) involves the formation of complexes including GATA2.

scholarly journals IMMUNE RESPONSES IN VITRO

1971 ◽  
Vol 134 (2) ◽  
pp. 395-416 ◽  
Author(s):  
Carl W. Pierce ◽  
Barbara M. Johnson ◽  
Harriet E. Gershon ◽  
Richard Asofsky
Keyword(s):  
Culture Conditions ◽  
Antibody Concentration ◽  
Spleen Cells ◽  
Immunoglobulin Class ◽  
Cell Densities ◽  
Erythrocyte Antigen ◽  
First Time ◽  
Kinetics Of

We have demonstrated for the first time that mouse spleen cells stimulated in vitro with heterologous erythrocytes developed immunoglobulin class-specific γM, γ1, γ2a+2b, and γA plaque-forming cell (PFC) responses. A modification of the hemolytic plaque technique, the addition of goat anti-mouse µ-chain antibody to the assay preparation, specifically prevented development of all γM PFC and enabled accurate and reproducible enumeration of immunoglobulin class-specific PFC after treatment with appropriate monospecific anti-globulins and complement. Culture conditions, with regard to medium, atmosphere, agitation, and spleen cell densities, were similar to those previously shown to support only γM PFC responses. Evaluation of the kinetics of appearance of PFC showed that γM PFC reached maximum numbers on days 4–5; the magnitude of this response was 3–10 times greater than γ1 γ2a+2b, or γA PFC which reached maximum numbers on days 5–6. Optimal erythrocyte antigen dose for γM PFC responses was 107/culture, whereas a dose of 106 erythrocytes/culture consistently stimulated optimal γ1 γ2a+2b, or γA PFC responses. Investigations of the effects of anti-erythrocyte antibody on γM and γG PFC responses indicated that antibody suppressed these responses by neutralizing the effective antigenic stimulus at the macrophage-dependent phase of the response. At the same antibody concentration, γG PFC responses were more effectively suppressed than γM PFC responses. Further, γG responses could be almost completely suppressed by antibody as long as 48 hr after initiation of cultures, whereas γM PFC responses could only be completely suppressed during the first 24 hr. These results were discusssed in terms of the role of antigen in the stimulation γM and γG antibody.

scholarly journals Activation of the G2/M-Specific Gene CLB2 Requires Multiple Cell Cycle Signals

2007 ◽  
Vol 27 (23) ◽  
pp. 8364-8373 ◽  
Author(s):  
J. Veis ◽  
H. Klug ◽  
M. Koranda ◽  
G. Ammerer
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Specific Gene ◽  
Histone H4 ◽  
Cycle Progression ◽  
Histone H4 Acetylation ◽  
Multiple Cell

ABSTRACT In budding yeast (Saccharomyces cerevisiae), the periodic expression of the G2/M-specific gene CLB2 depends on a DNA binding complex that mediates its repression during G1 and activation from the S phase to the exit of mitosis. The switch from low to high expression levels depends on the transcriptional activator Ndd1. We show that the inactivation of the Sin3 histone deacetylase complex bypasses the essential role of Ndd1 in cell cycle progression. Sin3 and its catalytic subunit Rpd3 associate with the CLB2 promoter during the G1 phase of the cell cycle. Both proteins dissociate from the promoter at the onset of the S phase and reassociate during G2 phase. Sin3 removal coincides with a transient increase in histone H4 acetylation followed by the expulsion of at least one nucleosome from the promoter region. Whereas the first step depends on Cdc28/Cln1 activity, Ndd1 function is required for the second step. Since the removal of Sin3 is independent of Ndd1 recruitment and Cdc28/Clb activity it represents a unique regulatory step which is distinct from transcriptional activation.

scholarly journals Molecular cloning of up-regulated cytoskeletal genes from regenerating skeletal muscle: potential role of myocyte enhancer factor 2 proteins in the activation of muscle-regeneration-associated genes

1997 ◽  
Vol 325 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Waleed M. AKKILA ◽  
Rebecca L. CHAMBERS ◽  
Olga I. ORNATSKY ◽  
John C. McDERMOTT
Keyword(s):  
Skeletal Muscle ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Cdna Clones ◽  
Myocyte Enhancer Factor 2 ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Adult Skeletal Muscle ◽  
Myocyte Enhancer Factor ◽  
Enhancer Factor

A subtractive hybridization and cloning strategy was used to identify genes that are up-regulated in regenerating compared with normal skeletal muscle. The gastrocnemius muscle of CD1 mice was injected with a myotoxic agent (BaCl2). A cDNA library was constructed from the regenerating muscle, and was screened with subtracted probes enriched in genes up-regulated during regeneration. Cofilin and vimentin cDNA clones were isolated. Both cofilin and vimentin were demonstrated to be overexpressed in regenerating compared with non-regenerating muscle (17-fold and 19-fold induction respectively). Cofilin and vimentin mRNAs also exhibited an increased expression in C2C12 myoblasts and a decreased expression in differentiated myotubes. Analysis of the regeneration-induced vimentin enhancer/promoter region revealed a consensus binding site for the myocyte enhancer factor 2 (MEF2) transcription factors. Electrophoretic mobility-shift assays and in vivo reporter assays revealed that MEF2 DNA-binding activity and transcriptional activation are increased in regenerating skeletal muscle, indicating that they may play a role in the activation of muscle genes during regeneration. These data suggest that both cofilin (an actin-regulatory protein) and vimentin (an intermediate filament) may be key components of the cytoskeletal reorganization that mediates muscle cell development and adult skeletal-muscle repair.

scholarly journals Protein Phosphatase 2A Reactivates FOXO3a through a Dynamic Interplay with 14-3-3 and AKT

2010 ◽  
Vol 21 (6) ◽  
pp. 1140-1152 ◽  
Author(s):  
Amrik Singh ◽  
Min Ye ◽  
Octavian Bucur ◽  
Shudong Zhu ◽  
Maria Tanya Santos ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Transcriptional Activation ◽  
Protein Phosphatase ◽  
Nuclear Translocation ◽  
Akt Phosphorylation ◽  
Forkhead Box ◽  
Phosphatase 2A ◽  
And Function ◽  
Dynamic Interplay

Forkhead box transcription factor FOXO3a, a key regulator of cell survival, is regulated by reversible phosphorylation and subcellular localization. Although the kinases regulating FOXO3a activity have been characterized, the role of protein phosphatases (PP) in the control of FOXO3a subcellular localization and function is unknown. In this study, we detected a robust interaction between FOXO3a and PP2A. We further demonstrate that 14-3-3, while not impeding the interaction between PP2A and FOXO3a, restrains its activity toward AKT phosphorylation sites T32/S253. Disruption of PP2A function revealed that after AKT inhibition, PP2A-mediated dephosphorylation of T32/S253 is required for dissociation of 14-3-3, nuclear translocation, and transcriptional activation of FOXO3a. Our findings reveal that distinct phosphatases dephosphorylate conserved AKT motifs within the FOXO family and that PP2A is entwined in a dynamic interplay with AKT and 14-3-3 to directly regulate FOXO3a subcellular localization and transcriptional activation.

scholarly journals Xihuang Pill Induces Apoptosis of Human Glioblastoma U-87 MG Cells via Targeting ROS-Mediated Akt/mTOR/FOXO1 Pathway

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Meng Shao ◽  
Zhenqiang He ◽  
Zhixin Yin ◽  
Peihong Ma ◽  
Qian Xiao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Translocation ◽  
Annexin V ◽  
Gas Chromatography Mass Spectrometry ◽  
Phase Cell ◽  
Caspase 9 ◽  
Cycle Arrest ◽  
First Time ◽  
Apoptotic Induction

Xihuang pill (XHP), a traditional Chinese herbal formula, has long been used as an effective agent against multiple tumors. The aim of this study is to evaluate the effects of XHP on the growth inhibition and apoptosis in glioblastoma U-87 MG cells. Gas chromatography-mass spectrometry (GC-MS) was performed for constituent analysis of XHP. Cell viability, cell cycle arrest, generation of reactive oxygen species (ROS), and apoptosis were measured by CCK-8 assay, PI/RNase staining, DCFH-DA assay, TUNEL assay, Annexin V-FITC/PI double staining, and JC-1 assay, respectively. The role of XHP in the regulation of Akt/mTOR/FOXO1 interaction was clarified by using Western Blotting (WB), immunofluorescence (IF), pharmacological inhibitor or antioxidant, and siRNA silencing. The results suggested that XHP could inhibit U-87 MG cells growth and arrest cells in S-phase cell cycle significantly and that the generation of ROS, collapse of mitochondrial membrane potential, enhancement of Bax/Bcl-xL ratio, and reduction of the precursor forms of caspase-9 and caspase-3 caused by XHP prompted that a ROS-mediated mitochondria-dependent apoptosis was possibly involved. Furthermore, XHP affected the Akt/mTOR/FOXO1 pathway via inhibiting the phosphorylation of Akt, mTOR, and FOXO1 and increasing both prototype and nuclear translocation of FOXO1. Inhibition of Akt, mTOR, and FOXO1 by specific inhibitors or siRNA could interpose the apoptotic induction. In conclusion, we demonstrate for the first time that XHP may regulate glioblastoma U-87 MG cell apoptosis via ROS-mediated Akt/mTOR/FOXO1 pathway.

scholarly journals Both E6 and E7 in HPV16 Promote the Glucose Uptake of GLUT1 in Lung Cancer Cells By Repressing NDRG2 Expression and Further Nuclear Translocation of β-Catenin

2021 ◽  
Author(s):  
Xin Wang ◽  
Ming-Zhe Wu ◽  
Na-Jin Gu ◽  
Shi-Yu Wang ◽  
Hong-Tao Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Glucose Uptake ◽  
Nuclear Translocation ◽  
Molecular Signaling ◽  
Genetic Manipulations ◽  
Ndrg2 Expression ◽  
E6 And E7 ◽  
E7 Proteins ◽  
First Time

Abstract Background: HPV16 is the most common infection subtype, among which E6 and E7 proteins are the most common carcinogenic proteins. Our previous studies found that E6 and E7 proteins regulated the expression of GLUT1 through multiple molecular signaling pathways in lung cancer. However, whether they can regulate the glucose uptake of GLUT1 and the underlying molecular mechanism has not been identified. Methods: The modulating effects of E6 or E7, NDRG2, β-catenin, and GLUT1 were detected by double directional genetic manipulations in lung cancer cell lines; The immunofluorescence was used to detect the effect of NDRG2 on the nuclear translocation of β-catenin; The glucose uptake level of GLUT1 was observed under the confocal microscope.Results: We demonstrated for the first time that E6 and E7 had inhibitory effects of NDRG2 which further resulted in increased β-catenin expression and promoted β-catenin nuclear translocation, furthermore promoted the expression and glucose uptake of GLUT1. Therefore, we hypothesized both E6 and E7 in HPV16 promoted the expression and glucose uptake of GLUT1 through HPV-NDRG2- β-catenin-GLUT1 axis. Conclusion: Our findings confirmed the regulatory role of tumor suppressor NDRG2 in the pathogenesis of lung cancer, and we further demonstrate the detail relationships among E6 and E7, NDRG2, β-catenin, and GLUT1; which provided a novel therapeutic target for tumor treatment.

scholarly journals Multi-parameter analysis of the kinetics of NF-κB signalling and transcription in single living cells

2002 ◽  
Vol 115 (6) ◽  
pp. 1137-1148 ◽  
Author(s):  
Glyn Nelson ◽  
Luminita Paraoan ◽  
David G. Spiller ◽  
Geraint J. C. Wilde ◽  
Mark A. Browne ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Fluorescent Proteins ◽  
Parameter Analysis ◽  
Nuclear Accumulation ◽  
Fusion Protein Expression ◽  
Kinetics Of

Proteins of the NF-κB transcription factor family normally reside in the cytoplasm of cells in a complex with IκB inhibitor proteins. Stimulation with TNFα leads to proteosomal degradation of the IκB proteins and nuclear translocation of the NF-κB proteins. Expression of p65 and IκBα fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IκBα-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression,the half-time of IκBα-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IκBα and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IκBα is the true substrate for TNFα stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor,leptomycin B (LMB), there was nuclear accumulation of IκBα-EGFP and p65-dsRed, with IκBα-EGFP accumulating more rapidly. No NF-κB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IκBα-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFα, degradation of IκBα-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-κB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-κB-dependent transcription occur even when nuclear export of NF-κB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

025. Prolactin signaling through the short form of its cognate receptor causes severe ovarian defect

2005 ◽  
Vol 17 (9) ◽  
pp. 68
Author(s):  
G. Gibori ◽  
J. Halpern ◽  
C. Stocco ◽  
P. Kelly ◽  
N. Binart
Keyword(s):  
Premature Ovarian Failure ◽  
Short Form ◽  
Ovarian Failure ◽  
Specific Gene ◽  
Steroidogenic Enzyme ◽  
Prolactin Signaling ◽  
Long Form ◽  
Membrane Bound ◽  
First Time

Extensive investigations from our laboratory have clarified the action and interaction of estradiol (E) and prolactin (PRL) on corpus luteum (CL) function. Our research has led us to discover and isolate a CL specific gene that encodes a protein we named PRAP, that associates with the intracellular domain of the short form (PRLRS) but not the long form (PRLRL) and whose expression is tightly regulated by E. Our laboratory and others have established that this protein, expressed in CL of every species investigated, is a novel 17 beta hydroxysteroid dehydrogenase (17bHSD-7) whose function is to catalyze the transformation of estrone to E. Our results with cells expressing only PRLRS revealed that PRL acting through PRLRS leads to phosphorylation of PRAP/17bHSD-7 (PRAP/17b) by JAK2 establishing for the first time that a steroidogenic enzyme can be phosphorylated through its association with a membrane bound protein. The association of PRAP/17b with the PRLRS and its phosphorylation leads to its stabilization. To further investigate the role of PRL signaling through PRLRS, we used PRLR(–/–) mice expressing the PRLRS as a transgene. The results obtained were totally unexpected and of great interest. The follicles of the ovaries, expressing PRLRS only, underwent premature development followed by severe granulosa and oocyte death leaving holes surrounding collapsed zona pellucida and premature ovarian failure. The observations that: (1) the expression of PRLRS in the ovaries of PRL null mice leads to inhibition in Foxo3a and of GALT, two proteins whose deletion/mutation causes similar premature ovarian failure; and (2) that GALT promoter activity is stimulated by Foxo3a transcription factor led us to hypothesize that PRL acting through PRLRS prevents the expression of Foxo3a, which normally stimulates GALT transcriptional activity. Absence of Foxo3a then leads to inhibition of GALT and increases in galactose and its metabolites, causing galactose toxicity and granulosa as well as oocyte cell death.

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