scholarly journals PGE2stimulates human brain natriuretic peptide expression via EP4and p42/44 MAPK

2006 ◽  
Vol 290 (5) ◽  
pp. H1740-H1746 ◽  
Author(s):  
Jian-Yong Qian ◽  
Alicia Leung ◽  
Pamela Harding ◽  
Margot C. LaPointe
Keyword(s):  
Protein Synthesis ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Prostaglandin E ◽  
Stimulation Of

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2(PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2increased hBNP promoter activity 3.5-fold. An EP4antagonist reduced the stimulatory effect of PGE2but not an EP1antagonist. Because EP4signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2stimulation of the hBNP promoter. H-89 at 5 μM decreased PGE2stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.

scholarly journals Isoproterenol and cAMP regulation of the human brain natriuretic peptide gene involves Src and Rac

2000 ◽  
Vol 278 (6) ◽  
pp. E1115-E1123 ◽  
Author(s):  
Quan He ◽  
Guiyun Wu ◽  
Margot C. Lapointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Kinase Inhibitor ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Luciferase Reporter Gene ◽  
Src Tyrosine Kinase

Brain natriuretic peptide (BNP) gene expression and chronic activation of the sympathetic nervous system are characteristics of the development of heart failure. We studied the role of the β-adrenergic signaling pathway in regulation of the human BNP (hBNP) promoter. An hBNP promoter (−1818 to +100) coupled to a luciferase reporter gene was transferred into neonatal cardiac myocytes, and luciferase activity was measured as an index of promoter activity. Isoproterenol (ISO), forskolin, and cAMP stimulated the promoter, and the β2-antagonist ICI 118,551 abrogated the effect of ISO. In contrast, the protein kinase A (PKA) inhibitor H-89 failed to block the action of cAMP and ISO. Pertussis toxin (PT), which inactivates Gαi, inhibited ISO- and cAMP-stimulated hBNP promoter activity. The Src tyrosine kinase inhibitor PP1 and a dominant-negative mutant of the small G protein Rac also abolished the effect of ISO and cAMP. Finally, we studied the involvement of M-CAT-like binding sites in basal and inducible regulation of the hBNP promoter. Mutation of these elements decreased basal and cAMP-induced activity. These data suggest that β-adrenergic regulation of hBNP is PKA independent, involves a Gαi-activated pathway, and targets regulatory elements in the proximal BNP promoter.

Phospholipase A 2 Metabolites Mediate Endothelin Stimulation of the Human Brain Natriuretic Peptide Promoter

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 721-721
Author(s):  
Quan He
Keyword(s):  
Gene Expression ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Phospholipase A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
P450 Monooxygenase ◽  
Stimulation Of

P155 Brain natriuretic peptide (BNP) gene expression accompanies cardiac hypertrophy and heart failure. The vasoconstrictor endothelin-1 (ET)may be involved in the development of these diseases. ET has also been shown to activate phospholipase A 2 (PLA 2 ). Thus we studied whether ET and PLA 2 metabolites regulate BNP gene expression. The hBNP promoter (-1818 to + 100) coupled to a luciferase reporter gene was transferred into neonatal ventricular myocytes (NVM),and luciferase activity was measured as an index of promoter activity. ET (10 -7 M)induced BNP mRNA in NVM as assessed by Northern blot. It also stimulated the hBNP promoter 4-fold vs control, an effect completely inhibited by actinomycin D. To test the involvement of different PLA 2 isoforms, transfected cells were treated with the Ca ++ -independent PLA 2 (iPLA 2 )inhibitor bromoenol lactone (BEL), the cytosolic PLA 2 inhibitor methyl arachidonyl fluorophosphonate, or the secretory PLA 2 inhibitor ONO-RS-082 prior to stimulation with ET. Only the iPLA 2 inhibitor BEL prevented ET-stimulated hBNP promoter activity. The PLA 2 metabolite lysophosphatidic acid (LPA) also activated the hBNP promoter (2.2-fold; n = 3), but lysophosphatidylcholine did not. To test whether arachidonic acid metabolites are involved in ET’s effect, cells were pretreated with either a lipoxygenase (LO), cyclooxygenase, or p450 monooxygenase inhibitor. Only the LO inhibitor baicalein prevented ET stimulation of the hBNP promoter. Finally, we studied the involvement of cis elements in ET-stimulated hBNP promoter activity. Deletion of BNP promoter sequences from -1818 to -408 and from -408 to -40 reduced ET’s effect by 54% and 78%, respectively. Moreover, ET-stimulated luciferase activity was reduced by 53% when the GATA element (at position -85 relative to the start site of transcription) was mutated. These data suggest that: 1) ET activates the hBNP promoter through a transcriptional mechanism; 2) LPA, perhaps generated by a BEL-sensitive iPLA 2 , is involved in ET’s effect; 3) a LO pathway may also mediate ET signaling; and 4) ET regulation of the hBNP promoter targets both distal and proximal cis elements, including GATA.

scholarly journals PKA, Rap1, ERK1/2, and p90RSK mediate PGE2 and EP4 signaling in neonatal ventricular myocytes

2010 ◽  
Vol 298 (1) ◽  
pp. H136-H143 ◽  
Author(s):  
Quan He ◽  
Pamela Harding ◽  
Margot C. LaPointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Growth Response ◽  
Ventricular Myocytes ◽  
Regulation Of Gene Expression ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dominant Negative Mutation ◽  
Stimulation Of

We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE2 production reduces hypertrophy after myocardial infarction in mice and 2) PGE2 acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE2 activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE2 activation of ERK1/2. PGE2 activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE2 regulation of gene expression. PGE2 stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE2 stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE2 regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos.

TNF-α induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes

2001 ◽  
Vol 280 (4) ◽  
pp. H1861-H1868 ◽  
Author(s):  
Eiji Hiraoka ◽  
Seinosuke Kawashima ◽  
Tomosaburo Takahashi ◽  
Yoshiyuki Rikitake ◽  
Tadahiro Kitamura ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cardiac Myocytes ◽  
Specific Inhibitor ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Dominant Negative Mutant ◽  
Tnf Α ◽  
Negative Mutant ◽  
Stimulation Of

The activation of phosphatidylinositol (PI) 3-kinase and Akt/protein kinase B (PKB) by tumor necrosis factor (TNF)-α and their roles on stimulation of protein synthesis were investigated in cultured neonatal rat cardiac myocytes. Treatment of cells with TNF-α resulted in enlargement of cell surface area and stimulation of protein synthesis without affecting myocyte viability. TNF-α induced marked activation of PI3-kinase and Akt/PKB, and the activation of PI3-kinase and Akt/PKB was rapid (maximal at 10 and 15 min, respectively) and concentration dependent. Akt/PKB activation by TNF-α was inhibited by a PI3-kinase-specific inhibitor LY-294002 and adenovirus-mediated expression of a dominant negative mutant of PI3-kinase, indicating that TNF-α activates Akt/PKB through PI3-kinase activation. Furthermore, TNF-α-induced protein synthesis was inhibited by pretreatment with LY-294002 and expression of a dominant negative mutant of PI3-kinase or Akt/PKB. These results indicate that activation of the PI3-kinase-Akt/PKB pathway plays an essential role in protein synthesis induced by TNF-α in cardiac myocytes.

Regulation of the human brain natriuretic peptide gene by GATA-4

2002 ◽  
Vol 283 (1) ◽  
pp. E50-E57 ◽  
Author(s):  
Quan He ◽  
Mariela Mendez ◽  
Margot C. LaPointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Nuclear Protein ◽  
Ventricular Myocytes ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression

Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. We previously showed that the human BNP (hBNP) proximal promoter region from −127 to −40 confers myocyte-specific expression. The proximal hBNP promoter contains several putative cis elements. Here we tested whether the proximal GATA element plays a role in basal and inducible regulation of the hBNP promoter. The hBNP promoter was coupled to a luciferase reporter gene (1818hBNPLuc) and transferred into neonatal ventricular myocytes (NVM), and luciferase activity was measured as an index of hBNP promoter activity. Mutation of the putative GATA element at −85 of the hBNP promoter [1818(mGATA)hBNPLuc] reduced activity by 97%. To study transactivation of the hBNP promoter, we co-transfected 1818hBNPLuc with the GATA-4 expression vector. GATA-4 activated 1818hBNPLuc, and this effect was eliminated by mutation of the proximal GATA element. Electrophoretic mobility shift assay showed that an oligonucleotide containing the hBNP GATA motif bound to cardiomyocyte nuclear protein, which was competed for by a consensus GATA oligonucleotide but not a mutated hBNP GATA element. The β-adrenergic agonist isoproterenol and its second messenger cAMP stimulated hBNP promoter activity and binding of nuclear protein to the proximal GATA element. Thus the GATA element in the proximal hBNP promoter is involved in both basal and inducible transcriptional regulation in cardiac myocytes.

Signaling mechanisms underlying strain-dependent brain natriuretic peptide gene transcription

2001 ◽  
Vol 79 (8) ◽  
pp. 640-645 ◽  
Author(s):  
Faquan Liang ◽  
Branka Kovacic-Milivojevic ◽  
Songcang Chen ◽  
Junfeng Cui ◽  
Fred Roediger ◽  
...  
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Cardiac Myocyte ◽  
Mechanical Strain ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Amino Terminal ◽  
Strain Dependent

Activation of brain natriuretic peptide (BNP) gene promoter activity represents one of the earliest and most reliable markers of ventricular cardiac myocyte hypertrophy. We recently demonstrated that mechanical strain increases immunoreactive BNP secretion, steady-state BNP mRNA levels and BNP gene transcriptional activity in neonatal rat myocyte cultures. We have also shown that strain-dependent BNP gene transcription is critically dependent on the functional integrity of a number of integrins (specfically β1, β3, and αvβ5 integrins) present on the surface of cardiac myocytes. When used alone, each of these antibodies resulted in a significant reduction in strain-dependent activation of a transfected hBNP-luciferase reporter and inhibition of a number of signaling pathways that have been linked to stimulation of this reporter (e.g., extracellular signal regulated kinase and c-Jun amino terminal kinase). The present study shows that combinations of these antibodies resulted in further reductions in hBNP gene promoter activity and inhibition of the relevant signaling cascades. These studies provide further support for the importance of integrin-matrix interactions in promoting strain-dependent changes in cardiac myocyte gene transcription.Key words: mechanical strain, brain natriuretic peptide, integrins, mitogen-activated protein kinase, cardiac myocyte.

scholarly journals RhoA Regulation of Cardiomyocyte Differentiation

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Mari Kaarbø ◽  
Denis I. Crane ◽  
Wayne G. Murrell
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Cardiomyocyte Differentiation ◽  
P19cl6 Cells ◽  
Heart Formation ◽  
Gene Organisation

Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiacα-actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes.

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

2021 ◽  
Author(s):  
Toshihiro Kobayashi ◽  
Hitomi Imachi ◽  
Kensaku Fukunaga ◽  
Jingya Lyu ◽  
Seisuke Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Promoter Activity ◽  
Active Form ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Dominant Negative ◽  
Activation Mechanism ◽  
Dominant Negative Mutant ◽  
Type I

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

scholarly journals β1-Integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal α-actin promoter in myoblasts

2000 ◽  
Vol 278 (6) ◽  
pp. H1736-H1743 ◽  
Author(s):  
Lei Wei ◽  
Wei Zhou ◽  
Lu Wang ◽  
Robert J. Schwartz
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Serum Response Factor ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Muscle Gene Expression ◽  
Actin Promoter ◽  
Muscle Gene ◽  
Negative Mutant

RhoA GTPase, a regulator of actin cytoskeleton, is also involved in regulating c- fos gene expression through its effect on serum response factor (SRF) transcriptional activity. We have also shown that RhoA plays a critical role in myogenesis and regulates expression of SRF-dependent muscle genes, including skeletal α-actin. In the present study, we examined whether the RhoA signaling pathway cross talks with other myogenic signaling pathways to modulate skeletal α-actin promoter activity in myoblasts. We found that extracellular matrix proteins and the β1-integrin stimulated RhoA-dependent activation of the α-actin promoter. The muscle-specific isoform β1Dselectively activated the α-actin promoter in concert with RhoA but inhibited the c- fos promoter. In addition, focal adhesion kinase (FAK) and phosphatidylinositol (PI) 3-kinase were required for full activation of the α-actin promoter by RhoA. Expression of a dominant negative mutant of FAK, application of wortmannin to cultured myoblasts, or expression of a dominant negative mutant of PI 3-kinase inhibited α-actin promoter activity induced by RhoA. These results suggest that RhoA, β1-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle gene expression.

NF-κB activation is required for the development of cardiac hypertrophy in vivo

2004 ◽  
Vol 287 (4) ◽  
pp. H1712-H1720 ◽  
Author(s):  
Yuehua Li ◽  
Tuanzhu Ha ◽  
Xiang Gao ◽  
Jim Kelley ◽  
David L. Williams ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Natriuretic Peptide ◽  
Weight Ratio ◽  
Dominant Negative ◽  
Heart Weight ◽  
Dominant Negative Mutant ◽  
Body Weight Ratio ◽  
Aortic Banding ◽  
Important Target

In the present study, we examined whether NF-κB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1–6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IκB-α dominant negative mutant (IκB-αM), a superrepressor of NF-κB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-κB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-κB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-β activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-κB activation in vivo by cardiac transfection of IκB-αM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-κB activity. Our results suggest that NF-κB activation is required for the development of cardiac hypertrophy in vivo and that NF-κB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.

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