Endothelin-1 induces phosphorylation of GATA-4 transcription factor in the HL-1 atrial-muscle cell line

2001 ◽  
Vol 359 (2) ◽  
pp. 375-380 ◽  
Author(s):  
Kazumi KITTA ◽  
Sophie A. CLÉMENT ◽  
Jane REMEIKA ◽  
Jeffrey B. BLUMBERG ◽  
Yuichiro J. SUZUKI
Keyword(s):  
Transcription Factor ◽  
Mapk Pathway ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Mobility Shift ◽  
Native Page ◽  
Endothelin 1 ◽  
Upward Shift ◽  
Atrial Muscle

The transcription factor GATA-4 plays a central role in the regulation of cardiac-muscle gene transcription. The present study demonstrates that endothelin-1 (ET-1) induces GATA-4 activation and phosphorylation. The treatment of HL-1 adult mouse atrial-muscle cells with ET-1 (30nM) caused a rapid increase in the DNA binding activity of GATA-4 within 3min. The activation was associated with an upward mobility shift of the GATA-4 band on native PAGE in an electrophoretic- mobility-shift assay. The upward shift of the GATA-4 band also occurred on SDS/PAGE as monitored by immunoblotting. The in vitro treatment of nuclear extracts with λ-protein phosphatase abolished the upward shift, indicating that GATA-4 was phosphorylated. ET-1 activated the p44/42 mitogen-activated protein kinase (MAPK) and the MAPK kinase (MEK) within 3min, and PD98059 (a specific inhibitor of MEK) abolished the ET-1-induced GATA-4 phosphorylation. PMA also caused the rapid activation of MAPK and the phosphorylation of GATA-4. In contrast, the activation of MAPK by phenylephrine or H2O2 was weak and did not lead to GATA-4 phosphorylation. Thus ET-1 induces a GATA-4 phosphorylation by activating a MEK–MAPK pathway.

scholarly journals Celecoxib as a Valuable Adjuvant in Cutaneous Melanoma Treated with Trametinib

2021 ◽  
Vol 22 (9) ◽  
pp. 4387
Author(s):  
Diana Valentina Tudor ◽  
Ioana Bâldea ◽  
Diana Elena Olteanu ◽  
Eva Fischer-Fodor ◽  
Virag Piroska ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Human Fibroblasts ◽  
In Vitro Study ◽  
Melanoma Cells ◽  
Mitogen Activated Protein Kinase ◽  
Culture Cells ◽  
Nm Range

Background: Melanoma patients stop responding to targeted therapies mainly due to mitogen activated protein kinase (MAPK) pathway re-activation, phosphoinositide 3 kinase/the mechanistic target of rapamycin (PI3K/mTOR) pathway activation or stromal cell influence. The future of melanoma treatment lies in combinational approaches. To address this, our in vitro study evaluated if lower concentrations of Celecoxib (IC50 in nM range) could still preserve the chemopreventive effect on melanoma cells treated with trametinib. Materials and Methods: All experiments were conducted on SK-MEL-28 human melanoma cells and BJ human fibroblasts, used as co-culture. Co-culture cells were subjected to a celecoxib and trametinib drug combination for 72 h. We focused on the evaluation of cell death mechanisms, melanogenesis, angiogenesis, inflammation and resistance pathways. Results: Low-dose celecoxib significantly enhanced the melanoma response to trametinib. The therapeutic combination reduced nuclear transcription factor (NF)–kB (p < 0.0001) and caspase-8/caspase-3 activation (p < 0.0001), inhibited microphthalmia transcription factor (MITF) and tyrosinase (p < 0.05) expression and strongly down-regulated the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway more significantly than the control or trametinib group (p < 0.0001). Conclusion: Low concentrations of celecoxib (IC50 in nM range) sufficed to exert antineoplastic capabilities and enhanced the therapeutic response of metastatic melanoma treated with trametinib.

scholarly journals Biliverdin Reductase, a Novel Regulator for Induction of Activating Transcription Factor-2 and Heme Oxygenase-1

2004 ◽  
Vol 279 (19) ◽  
pp. 19916-19923 ◽  
Author(s):  
Anatoliy Kravets ◽  
Zhenbo Hu ◽  
Tihomir Miralem ◽  
Michael D. Torno ◽  
Mahin D. Maines
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Mapk Pathway ◽  
Regulation Of Gene Expression ◽  
Bzip Transcription Factor ◽  
Heme Oxygenase 1 ◽  
Activity Levels ◽  
Mobility Shift ◽  
Unlabeled Probe

Biliverdin IXα reductase (BVR) catalyzes reduction of the HO activity product, biliverdin, to bilirubin. hBVR is a serine/threonine kinase that contains a bZip domain. Presently, regulation of gene expression by hBVR was examined. 293A cells were infected with adenovirusdoxycycline (Ad-Dox)-inducible hBVR cDNA. High level expression of hBVR was determined at mRNA, protein, and activity levels 8 h after induction. Cell signal transduction microarray analysis of cells infected with expression or with the control Ad-inverted (INV)-hBVR vector identifiedATF-2among several up-regulated genes. ATF-2 is a bZip transcription factor for activation of cAMP response element (CRE) and a dimeric partner to c-junin MAPK pathway that regulates the stress protein, HO-1, expression. Northern and Western blot analyses showed increases of ∼10-fold in ATF-2 mRNA and protein at 16 and 24 h after Dox addition. Ad-INV-hBVR did not effect ATF-2 expression. In hBVR-infected cells, levels of HO-1 mRNA and protein were increased.In vitrotranslated hBVR and nuclear extract containing hBVR in gel mobility-shift assay bound to AP-1 sites in theATF-2promoter region and to an oligonucleotide containing the CRE site. Both bindings could be competed out by excess unlabeled probe; in the presence of hBVR antibody, they displayed shifted bands. Co-transfection of hBVR withATF-2or c-junpromoters caused a severalfold increase in luciferase activity. hBVR modulation ofATF-2andHO-1expression suggests it has a potential role in regulation of AP-1 and cAMP-regulated genes and a role in cell signaling. We propose that increased expression of the protein can be used to alter the gene expression profile in the cell.

scholarly journals Thymoquinone, a Dietary Bioactive Compound, Exerts Anti-Inflammatory Effects in Colitis by Stimulating Expression of the Colonic Epithelial PPAR-γ Transcription Factor

Nutrients ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 1343
Author(s):  
Balaji Venkataraman ◽  
Saeeda Almarzooqi ◽  
Vishnu Raj ◽  
Abdullah T. Alhassani ◽  
Ahmad S. Alhassani ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Activity Index ◽  
Nigella Sativa ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Ppar Γ ◽  
Anti Inflammatory ◽  
Ht 29

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders with increasing incidence and prevalence worldwide. Here, we investigated thymoquinone (TQ), a naturally occurring phytochemical present in Nigella sativa, for anti-inflammatory effects in colonic inflammation. To address this, we used in vivo (mice) and in vitro (HT-29 cells) models in this investigation. Our results showed that TQ treatment significantly reduced the disease activity index (DAI), myeloperoxidase (MPO) activity, and protected colon microscopic architecture. In addition, TQ also reduced the expression of proinflammatory cytokines and mediators at both the mRNA and protein levels. Further, TQ decreased phosphorylation of the activated mitogen-activated protein kinase (MAPK) signaling pathway and nuclear factor kappa B (NF-κB) proteins and enhanced colon epithelial PPAR-γ transcription factor expression. TQ significantly decreased proinflammatory chemokines (CXCL-1 and IL-8), and mediator (COX-2) mRNA expression in HT-29 cells treated with TNF-α. TQ also increased HT-29 PPAR-γ mRNA, PPAR-γ protein expression, and PPAR-γ promoter activity. These results indicate that TQ inhibits MAPK and NF-κB signaling pathways and transcriptionally regulates PPAR-γ expression to induce potent anti-inflammatory activity in vivo and in vitro models of colon inflammation.

scholarly journals Role for the Ran Binding Protein, Mog1p, in Saccharomyces cerevisiae SLN1-SKN7 Signal Transduction

2004 ◽  
Vol 3 (6) ◽  
pp. 1544-1556 ◽  
Author(s):  
Jade Mei-Yeh Lu ◽  
Robert J. Deschenes ◽  
Jan S. Fassler
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
Osmotic Stress ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Osmotic Response ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

ABSTRACT Yeast Sln1p is an osmotic stress sensor with histidine kinase activity. Modulation of Sln1 kinase activity in response to changes in the osmotic environment regulates the activity of the osmotic response mitogen-activated protein kinase pathway and the activity of the Skn7p transcription factor, both important for adaptation to changing osmotic stress conditions. Many aspects of Sln1 function, such as how kinase activity is regulated to allow a rapid response to the continually changing osmotic environment, are not understood. To gain insight into Sln1p function, we conducted a two-hybrid screen to identify interactors. Mog1p, a protein that interacts with the yeast Ran1 homolog, Gsp1p, was identified in this screen. The interaction with Mog1p was characterized in vitro, and its importance was assessed in vivo. mog1 mutants exhibit defects in SLN1-SKN7 signal transduction and mislocalization of the Skn7p transcription factor. The requirement for Mog1p in normal localization of Skn7p to the nucleus does not fully account for the mog1-related defects in SLN1-SKN7 signal transduction, raising the possibility that Mog1p may play a role in Skn7 binding and activation of osmotic response genes.

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

scholarly journals Inhibition of Epithelial CC-Family Chemokine Synthesis by the Synthetic Chalcone DMPF-1 via Disruption of NF-κB Nuclear Translocation and Suppression of Experimental Asthma in Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-15 ◽  
Author(s):  
Revathee Rajajendram ◽  
Chau Ling Tham ◽  
Mohamad Nadeem Akhtar ◽  
Mohd Roslan Sulaiman ◽  
Daud Ahmad Israf
Keyword(s):  
Airway Hyperresponsiveness ◽  
Nuclear Translocation ◽  
Mapk Pathway ◽  
Pulmonary Inflammation ◽  
Mitogen Activated Protein Kinase ◽  
Epithelial Cell Line ◽  
Cell Hyperplasia ◽  
Mitogen Activated Protein ◽  
Cc Chemokines

Asthma is associated with increased pulmonary inflammation and airway hyperresponsiveness. The interaction between airway epithelium and inflammatory mediators plays a key role in the pathogenesis of asthma.In vitrostudies evaluated the inhibitory effects of 3-(2,5-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one (DMPF-1), a synthetic chalcone analogue, upon inflammation in the A549 lung epithelial cell line. DMPF-1 selectively inhibited TNF-α-stimulated CC chemokine secretion (RANTES, eotaxin-1, and MCP-1) without any effect upon CXC chemokine (GRO-αand IL-8) secretion. Western blot analysis further demonstrated that the inhibitory activity resulted from disruption of p65NF-κB nuclear translocation without any effects on the mitogen-activated protein kinase (MAPK) pathway. Treatment of ovalbumin-sensitized and ovalbumin-challenged BALB/c mice with DMPF-1 (0.2–100 mg/kg) demonstrated significant reduction in the secretion and gene expression of CC chemokines (RANTES, eotaxin-1, and MCP-1) and Th2 cytokines (IL-4, IL-5, and IL-13). Furthermore, DMPF-1 treatment inhibited eosinophilia, goblet cell hyperplasia, peripheral blood total IgE, and airway hyperresponsiveness in ovalbumin-sensitized and ovalbumin-challenged mice. In conclusion, these findings demonstrate the potential of DMPF-1, a nonsteroidal compound, as an antiasthmatic agent for further pharmacological evaluation.

scholarly journals Transient Silencing of a Type IV P-Type ATPase,Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
S. E. Hurst ◽  
S. C. Minkin ◽  
J. Biggerstaff ◽  
M. S. Dhar
Keyword(s):  
Type 2 Diabetes ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Mapk Pathway ◽  
Specific Inhibitor ◽  
C2c12 Myotubes ◽  
Glucose Transporter 1 ◽  
Transfected Cells

Atp10cis a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C,Atp10cexpression was alteredin vitroin C2C12 skeletal muscle myotubes by transient transfection with anAtp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and p44/42. Additionally, glucose transporter-1 (GLUT1) was significantly upregulated; no changes in glucose transporter-4 (GLUT4) expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate thatAtp10cregulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

scholarly journals Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf

1995 ◽  
Vol 311 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M A Bevilacqua ◽  
M C Faniello ◽  
P D′Agostino ◽  
B Quaresima ◽  
M T Tiano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mobility Shift ◽  
Differentiated Cells ◽  
Binding Factor ◽  
H Gene ◽  
Ferritin Gene ◽  
Mobility Shift Assay ◽  
Promoter Interaction

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

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