scholarly journals Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes

2010 ◽  
Vol 119 (7) ◽  
pp. 273-282 ◽  
Author(s):  
Chiung-Zuan Chiu ◽  
Bao-Wei Wang ◽  
Tun-Hui Chung ◽  
Kou-Gi Shyu
Keyword(s):  
Angiotensin Ii ◽  
Serum Response Factor ◽  
Signal Transduction Pathway ◽  
Luciferase Assay ◽  
Cardiomyocyte Hypertrophy ◽  
Erk Pathway ◽  
Mobility Shift ◽  
Hypoxic Injury ◽  
Neonatal Cardiomyocytes ◽  
Mobility Shift Assay

Hypoxic injury to cardiomyocytes is a stress that causes cardiac pathology through cardiac-restricted gene expression. SRF (serum-response factor) and myocardin are important for cardiomyocyte growth and differentiation in response to myocardial injuries. Previous studies have indicated that AngII (angiotensin II) stimulates both myocardin expression and cardiomyocyte hypertrophy. In the present study, we evaluated the expression of myocardin and AngII after hypoxia in regulating gene transcription in neonatal cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and AngII were evaluated. Different signal transduction pathway inhibitors were used to identify the pathway(s) responsible for myocardin expression. An EMSA (electrophoretic mobility-shift assay) was used to identify myocardin/SRF binding, and a luciferase assay was used to identify transcriptional activity of myocardin/SRF in neonatal cardiomyocytes. Both myocardin and AngII expression increased after hypoxia, with AngII appearing at an earlier time point than myocardin. Myocardin expression was stimulated by AngII and ERK (extracellular-signal-regulated kinase) phosphorylation, but was suppressed by an ARB (AngII type 1 receptor blocker), an ERK pathway inhibitor and myocardin siRNA (small interfering RNA). AngII increased both myocardin expression and transcription in neonatal cardiomyocytes. Binding of myocardin/SRF was identified using an EMSA, and a luciferase assay indicated the transcription of myocardin/SRF in neonatal cardiomyocytes. Increased BNP (B-type natriuretic peptide), MHC (myosin heavy chain) and [3H]proline incorporation into cardiomyocytes was identified after hypoxia with the presence of myocardin in hypertrophic cardiomyocytes. In conclusion, hypoxia in cardiomyocytes increased myocardin expression, which is mediated by the induction of AngII and the ERK pathway, to cause cardiomyocyte hypertrophy. Myocardial hypertrophy was identified as an increase in transcriptional activities, elevated hypertrophic and cardiomyocyte phenotype markers, and morphological hypertrophic changes in cardiomyocytes.

scholarly journals Angiotensin II and the JNK pathway mediate urotensin II expression in response to hypoxia in rat cardiomyocytes

2014 ◽  
Vol 220 (3) ◽  
pp. 233-246 ◽  
Author(s):  
Chiung-Zuan Chiu ◽  
Bao-Wei Wang ◽  
Kou-Gi Shyu
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Collagen I ◽  
Neonatal Rat ◽  
Luciferase Assay ◽  
Cardiomyocyte Hypertrophy ◽  
Urotensin Ii ◽  
Jnk Pathway ◽  
Rat Cardiomyocytes ◽  
Neonatal Cardiomyocytes

Cardiomyocyte hypoxia causes cardiac hypertrophy through cardiac-restricted gene expression. Urotensin II (UII) cooperates with activating protein 1 (AP1) to regulate cardiomyocyte growth in response to myocardial injuries. Angiotensin II (AngII) stimulates UII expression, reactive oxygen species (ROS) production, and cardiac hypertrophy. This study aimed to evaluate the expression of UII, ROS, and AngII as well as their genetic transcription after hypoxia treatment in neonatal cardiomyocytes. Cultured neonatal rat cardiomyocytes were subjected to hypoxia for different time periods. UII (Uts2) protein levels increased after 2.5% hypoxia for 4 h with earlier expression of AngII and ROS. Both hypoxia and exogenously added AngII or Dp44mT under normoxia stimulated UII expression, whereas AngII receptor blockers, JNK inhibitors (SP600125), JNK siRNA, or N-acetyl-l-cysteine (NAC) suppressed UII expression. The gel shift assay indicated that hypoxia induced an increase in DNA–protein binding between UII and AP1. The luciferase assay confirmed an increase in transcription activity of AP1 to the UII promoter under hypoxia. After hypoxia, an increase in 3H-proline incorporation in the cardiomyocytes and expression of myosin heavy chain protein, indicative of cardiomyocyte hypertrophy, were observed. In addition, hypoxia increased collagen I expression, which was inhibited by SP600125, NAC, and UII siRNA. In summary, hypoxia in cardiomyocytes increases UII and collagen I expression through the induction of AngII, ROS, and the JNK pathway causing cardiomyocyte hypertrophy and fibrosis.

scholarly journals A Functional SNP in the Promoter of LBX1 Is Associated With the Development of Adolescent Idiopathic Scoliosis Through Involvement in the Myogenesis of Paraspinal Muscles

2021 ◽  
Vol 9 ◽  
Author(s):  
Leilei Xu ◽  
Zhenhua Feng ◽  
Zhicheng Dai ◽  
Wayne Y. W. Lee ◽  
Zhichong Wu ◽  
...  
Keyword(s):  
Adolescent Idiopathic Scoliosis ◽  
Idiopathic Scoliosis ◽  
Muscle Development ◽  
Luciferase Assay ◽  
Paraspinal Muscles ◽  
Mobility Shift ◽  
Putative Promoter Region ◽  
Mobility Shift Assay ◽  
First Time ◽  
Functional Snp

Previous studies have shown that LBX1 is associated with adolescent idiopathic scoliosis (AIS) in multiple populations. For the first time, rs1322330 located in the putative promoter region of LBX1 was found significantly associated with AIS in the Chinese population [p = 6.08 × 10–14, odds ratio (OR) = 1.42, 95% confidence interval of 1.03–1.55]. Moreover, the luciferase assay and electrophoretic mobility shift assay supported that the allele A of rs1322330 could down-regulate the expression of LBX1 in the paraspinal muscles of AIS. In addition, silencing LBX1 in the myosatellite cells resulted in significantly inhibited cell viability and myotube formation, which supported an essential role of LBX1 in muscle development of AIS. To summarize, rs1322330 may be a novel functional SNP regulating the expression of LBX1, which was involved in the etiology of AIS possibly via regulation of myogenesis in the paraspinal muscles.

Signal Transducer and Activator of Transcription (Stat)-3 Activates the Receptor Tyrosine Kinase-Like Orphan (ROR)-1 Gene in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 57-57
Author(s):  
Li Ping ◽  
David Harris ◽  
Zhiming Liu ◽  
Michael Keating ◽  
Zeev Estrov
Keyword(s):  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Luciferase Assay ◽  
Mobility Shift ◽  
Protein Levels ◽  
Cell Chip ◽  
Stat3 Phosphorylation ◽  
Mobility Shift Assay ◽  
Activation Sequence ◽  
Gene Mrna

Abstract Abstract 57 ROR1, an embryonic protein involved in organogenesis and Wnt signaling, is expressed in B-cell CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors ψ-interferon activation sequence (GAS)-like elements typically activated by Stat3, we sought to determine whether Stat3 activates ROR1. In MM1 cells interleukin (IL)-6 induced Stat3 phosphorylation and upregulated ROR1 whereas STAT3-siRNA downregulated both Stat3 and ROR1 protein levels, suggesting that Stat3 transcribes ROR1. Therefore, we cloned the human ROR1 promoter, generated a series of truncated promoter constructs and assessed their activity by using the luciferase assay. We found that IL-6 augmented the luciferase activity of ROR1 -195, ROR1 -666, ROR1 -834, and co-transfection with Stat3-siRNA significantly attenuated it, suggesting that IL-6 enhanced ROR1 expression by activating Stat3. Furthermore, we established that a region, located between bp -122 and -134, harbors a GAS-like element and activates the ROR1 promoter upon exposure to IL-6. Binding of Stat3 to that region in IL-6-stimulated MM1 cells was confirmed by the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether Stat3 transcribes ROR1 in CLL, we obtained fresh CLL cells and by using the same GAS-like element-containing probe we performed EMSA. CLL cell nuclear protein bound this probe and anti-Stat3 and -phsophoserine Stat3 antibodies induced a super-shift. CLL cell ChIP confirmed that Stat3 binds to the promoter of ROR1 as well as the promoters of the Stat3-regulated genes STAT3, c-Myc and P21, but not that of the control gene RPL30. Finally, using qRT-PCR and western immunoblotting we determined that STAT3-shRNA downregulated ROR1, STAT3 and STAT3-regulated gene mRNA by 4-6 fold, and Stat3 and ROR1 protein levels by 50%. Taken together, these data suggest that constitutively activated Stat3 binds to the ROR1 promoter, activates transcription, and induces production of ROR1 protein in CLL cells. Disclosures: No relevant conflicts of interest to declare.

Heat shock treatment suppresses angiotensin II-induced activation of NF-κB pathway and heart inflammation: a role for IKK depletion by heat shock?

2004 ◽  
Vol 287 (3) ◽  
pp. H1104-H1114 ◽  
Author(s):  
Yu Chen ◽  
André-Patrick Arrigo ◽  
R. William Currie
Keyword(s):  
Angiotensin Ii ◽  
Heat Shock ◽  
Nuclear Translocation ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Mobility Shift ◽  
Iκb Kinase ◽  
Sprague Dawley Rats ◽  
Mobility Shift Assay ◽  
High Level

Heat shock (HS) proteins (Hsps) function in tissue protection through their chaperone activity and by interacting with cell signaling pathways to suppress apoptosis. Here, we investigated the effect of HS treatment on the nuclear factor (NF)-κB signaling pathway in the angiotensin II (ANG II) model of inflammation. Male Sprague-Dawley rats were divided into sham and HS-, ANG II-, and HS + ANG II-treated groups. HS treatment was administered 24 h before the initiation of ANG II infusion. HS treatment (42°C for 15 min) decreased 7-day ANG II-induced hypertension from 191 ± 4 to 147 ± 3 mmHg ( P < 0.01). Histological staining of hearts showed that HS treatment reduced ANG II-induced leukocyte infiltration, perivascular and interstitial inflammation, and fibrosis. Heart NF-κB nuclear translocation and activity, examined by Western blot analysis and electrophoretic mobility shift assay, was suppressed by HS treatment. HS treatment depleted IκB kinase-α (IKK-α) and phosphorylated IKK-α and suppressed the depletion of IκB-α and the accumulation of phosphorylated IκB-α. HS treatment blocked ANG II induced expression of IL-6 and ICAM-1 in the heart. ANG II and HS treatment induced high-level expression of Hsp27 and Hsp70 and their phosphorylation. Phosphorylated isoforms of Hsp27 and Hsp70 may play an important role in protecting the heart against ANG II-induced inflammation.

scholarly journals Sodium tanshinone IIA sulfonate depresses angiotensin II-induced cardiomyocyte hypertrophy through MEK/ERK pathway

2007 ◽  
Vol 39 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Le Yang ◽  
Xiaojing Zou ◽  
Qiansheng Liang ◽  
Hao Chen ◽  
Jun Feng ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Tanshinone Iia ◽  
Cardiomyocyte Hypertrophy ◽  
Erk Pathway ◽  
Sodium Tanshinone Iia Sulfonate

scholarly journals Dysbindin is a potent inducer of RhoA–SRF-mediated cardiomyocyte hypertrophy

2013 ◽  
Vol 203 (4) ◽  
pp. 643-656 ◽  
Author(s):  
Ashraf Yusuf Rangrez ◽  
Alexander Bernt ◽  
Reza Poyanmehr ◽  
Violetta Harazin ◽  
Inka Boomgaarden ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Signaling Pathways ◽  
Serum Response Factor ◽  
Susceptibility Gene ◽  
In Vivo Studies ◽  
Luciferase Assay ◽  
Cardiomyocyte Hypertrophy ◽  
Potent Inducer ◽  
Binding Partner

Dysbindin is an established schizophrenia susceptibility gene thoroughly studied in the context of the brain. We have previously shown through a yeast two-hybrid screen that it is also a cardiac binding partner of the intercalated disc protein Myozap. Because Dysbindin is highly expressed in the heart, we aimed here at deciphering its cardiac function. Using a serum response factor (SRF) response element reporter-driven luciferase assay, we identified a robust activation of SRF signaling by Dysbindin overexpression that was associated with significant up-regulation of SRF gene targets, such as Acta1 and Actc1. Concurrently, we identified RhoA as a novel binding partner of Dysbindin. Further phenotypic and mechanistic characterization revealed that Dysbindin induced cardiac hypertrophy via RhoA–SRF and MEK1–ERK1 signaling pathways. In conclusion, we show a novel cardiac role of Dysbindin in the activation of RhoA–SRF and MEK1–ERK1 signaling pathways and in the induction of cardiac hypertrophy. Future in vivo studies should examine the significance of Dysbindin in cardiomyopathy.

scholarly journals The R2R3-type MYB transcription factor MdMYB90-like is responsible for the enhanced skin color of an apple bud sport mutant

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Chao Sun ◽  
Chunming Wang ◽  
Wang Zhang ◽  
Shuai Liu ◽  
Weiyao Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Skin Color ◽  
Anthocyanin Biosynthesis ◽  
Regulatory Gene ◽  
Gene Promoter ◽  
Luciferase Assay ◽  
Myb Transcription Factor ◽  
Anthocyanin Content ◽  
Mobility Shift ◽  
Mobility Shift Assay

AbstractThe anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes (MdCHS and MdUFGT) and other transcription factor genes (MdMYB1 and MdbHLH3) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3, and MdMYB1. The mutant had reduced levels of DNA methylation in two regions (−1183 to −988 and −2018 to −1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.

scholarly journals Association between polymorphism in the promoter region of lncRNA GAS5 and the risk of colorectal cancer

2019 ◽  
Vol 39 (4) ◽  
Author(s):  
Yajie Wang ◽  
Shenshen Wu ◽  
Xi Yang ◽  
Xiaobo Li ◽  
Rui Chen
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Promoter Region ◽  
Expression Level ◽  
Luciferase Assay ◽  
Migration And Invasion ◽  
Mobility Shift ◽  
Dual Luciferase Assay ◽  
Invasion And Migration ◽  
Mobility Shift Assay

AbstractThe growth arrest special 5 (GAS5), as a research hotspot of long noncoding RNAs (lncRNAs), has been reported to be associated with colorectal cancer (CRC). However, the association between polymorphisms in GAS5 and the risk of CRC was not clear. In the present study, a case–control study in 1078 CRC patients and 1175 matched healthy controls was performed to evaluate the association between the potential functional genetic variants in GAS5 and the risk of CRC. PCR-TaqMan, qPCR, dual-luciferase assay, electrophoretic mobility shift assay (EMSA), flow cytometry, migration and invasion assays were performed to evaluate the function of polymorphism. Results showed that subjects carrying the rs55829688 CT/TT genotypes had a significantly higher risk of CRC when compared with the CC genotype. Further qPCR assay confirmed that the CRC tissues with rs55829688 CT/TT genotypes had a higher GAS5 mRNA expression level. The dual-luciferase assay, qPCR and EMSA assay revealed that rs55829688 T>C polymorphism could decrease the expression level of GAS5 by impacting the binding ability of the transcription factor Yin Yang-1 (YY1) to the GAS5 promoter region. The expression of apoptosis-related proteins were detected by Western blot. Further, flow cytometry, migration, and invasion experiments showed that GAS5 repressed apoptosis and increased invasion and migration capability of CRC cells. Taken together, our findings provided evidence that the rs55829688 variant in the GAS5 promoter was associated with the risk of CRC and decreased expression of GAS5 by affecting the binding affinity of the transcription factors YY1 to GAS5.

scholarly journals MiR-27a-3p/Hoxa10 Axis Regulates Angiotensin II-Induced Cardiomyocyte Hypertrophy by Targeting Kv4.3 Expression

2021 ◽  
Vol 12 ◽  
Author(s):  
Xuefeng Cao ◽  
Zheng Zhang ◽  
Yu Wang ◽  
Weichao Shan ◽  
Ruiting Wang ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Myocardial Hypertrophy ◽  
Pathological Process ◽  
Luciferase Assay ◽  
Cardiomyocyte Hypertrophy ◽  
Electrical Remodeling ◽  
Transcriptional Level ◽  
Ang Ii ◽  
Clinical Prevention

Cardiac hypertrophy is a common pathological process of various cardiovascular diseases, which is often accompanied with structural and electrical remodeling, and can even lead to sudden cardiac death. However, its molecular mechanism still remains largely unknown. Here, we induced cardiomyocyte hypertrophy by angiotensin II (Ang II), and found that miR-27a-3p and hypertrophy-related genes were up-regulated. Further studies showed that miR-27a-3p-inhibitor can alleviate myocardial hypertrophy and electrical remodeling. Moreover, luciferase assay confirmed that miR-27a-3p could regulate the expression of downstream Hoxa10 at the transcriptional level by targeting at its 3′UTR. At the same time, the protein expression of Hoxa10 was significantly reduced in Ang II-treated cardiomyocytes. Furthermore, overexpression of Hoxa10 can reverse myocardial hypertrophy and electrical remodeling induced by Ang II in cardiomyocytes. Finally, we found that Hoxa10 positively regulated the expression of potassium channel protein Kv4.3 which was down-regulated in hypertrophic cardiomyocytes. Taken together, our results revealed miR-27a-3p/Hoxa10/Kv4.3 axis as a new mechanism of Ang II-induced cardiomyocyte hypertrophy, which provided a new target for clinical prevention and treatment of cardiac hypertrophy and heart failure.

scholarly journals Rap1GAP Mediates Angiotensin II-Induced Cardiomyocyte Hypertrophy by Inhibiting Autophagy and Increasing Oxidative Stress

2021 ◽  
Vol 2021 ◽  
pp. 1-20
Author(s):  
Yan Gao ◽  
Di Zhao ◽  
Wen-zhi Xie ◽  
Tingting Meng ◽  
Chunxiao Xu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heart Failure ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Cardiomyocyte Hypertrophy ◽  
Ros Generation ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Neonatal Cardiomyocytes ◽  
And Oxidative Stress

Abnormal autophagy and oxidative stress contribute to angiotensin II- (Ang II-) induced cardiac hypertrophy and heart failure. We previously showed that Ang II increased Rap1GAP gene expression in cardiomyocytes associated with hypertrophy and autophagy disorders. Using real-time PCR and Western blot, we found that Rap1GAP expression was increased in the heart of Sprague Dawley (SD) rats infused by Ang II compared with saline infusion and in Ang II vs. vehicle-treated rat neonatal cardiomyocytes. Overexpression of Rap1GAP in cultured cardiomyocytes exacerbated Ang II-induced cardiomyocyte hypertrophy, reactive oxygen species (ROS) generation, and cell apoptosis and inhibited autophagy. The increased oxidative stress caused by Rap1GAP overexpression was inhibited by the treatment of autophagy agonists. Knockdown of Rap1GAP by siRNA markedly attenuated Ang II-induced cardiomyocyte hypertrophy and oxidative stress and enhanced autophagy. The AMPK/AKT/mTOR signaling pathway was inhibited by overexpression of Rap1GAP and activated by the knockdown of Rap1GAP. These results show that Rap1GAP-mediated pathway might be a new mechanism of Ang II-induced cardiomyocyte hypertrophy, which could be a potential target for the future treatment of cardiac hypertrophy and heart failure.

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