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

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00904.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. H1740-H1746 ◽

Cited By ~ 22

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.

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

Hypertension ◽

10.1161/hyp.36.suppl_1.721-b ◽

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.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00274.2001 ◽

2002 ◽

Vol 283 (1) ◽

pp. E50-E57 ◽

Cited By ~ 26

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.

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Signaling mechanisms underlying strain-dependent brain natriuretic peptide gene transcription

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y01-036 ◽

2001 ◽

Vol 79 (8) ◽

pp. 640-645 ◽

Cited By ~ 8

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.

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RhoA Regulation of Cardiomyocyte Differentiation

The Scientific World JOURNAL ◽

10.1155/2013/491546 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 1

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00251.2009 ◽

2010 ◽

Vol 298 (1) ◽

pp. H136-H143 ◽

Cited By ~ 25

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.

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Kv1.5 potassium channel gene regulation by Sp1 transcription factor and oxidative stress

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00637.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. H2719-H2725 ◽

Cited By ~ 22

Author(s):

Samuel J. Fountain ◽

Alex Cheong ◽

Jing Li ◽

Naciye Y. Dondas ◽

Fanning Zeng ◽

...

Keyword(s):

Oxidative Stress ◽

Transcription Factor ◽

Smooth Muscle ◽

Potassium Channel ◽

Promoter Activity ◽

Dominant Negative ◽

Luciferase Reporter ◽

Therapeutic Drug ◽

Luciferase Reporter Gene ◽

Sp1 Transcription Factor

KV1.5, a voltage-gated potassium channel, has functional importance in regulating blood vessel tone and cardiac action potentials and is a target for numerous therapeutic drug development programs. Despite the importance of KV1.5, there is little knowledge of the mechanisms controlling expression of its underlying gene, Kcna5. We identified a 5′ flanking region of the murine Kcna5 gene that drives expression of a luciferase reporter gene in primary smooth muscle cells and a smooth muscle cell line. The promoter contained CACCC nucleotide motifs, which we have shown to bind the Sp1 transcription factor in the aorta under physiological conditions in vivo. Inhibition of Sp1- Kcna5 promoter interactions using mithramycin A, a dominant-negative Sp1 mutant, or disruption of the CACCC boxes by mutagenesis inhibited promoter activity. Conversely, expression of exogenous Sp1 augmented promoter activity. Sp1 has known sensitivity to oxidative stress and, consistent with this property, Kcna5 promoter activity was suppressed by hydrogen peroxide-induced oxidative stress. Our results show that Kcna5 promoter activity in vascular smooth muscle is critically dependent on Sp1 regulation via CACCC box motifs and identify mechanisms that potentially influence the expression of KV1.5 channel expression in physiological or pathological conditions.

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Unusual 5'-regulatory structure and regulation of the murine Mlc1 gene: Lack of promoter-specific functional elements

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2011.2314 ◽

2011 ◽

Vol 2 (1) ◽

pp. 11

Author(s):

Darja Henseler ◽

Jonathan D. Turner ◽

Matthias Eckhardt ◽

Maaike Van der Mark ◽

Yanina Revsin ◽

...

Keyword(s):

Reporter Gene ◽

In Silico ◽

Promoter Activity ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Regulatory Structure ◽

Luciferase Reporter Gene ◽

Promoter Regions ◽

Potential Promoter

The MLC1 gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC), which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly). Variants of MLC1 have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1). Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine Mlc1 with respect to regulatory elements for gene expression. A promoter search and an in silico analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity in vitro. We found two alternative first exons for the murine Mlc1 but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential cis-acting elements far outside of the region. In silico analysis indicated an uncommon promoter structure for Mlc1, with CCAAT-boxes representing the only noticeable elements.

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Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale

International Journal of Molecular Sciences ◽

10.3390/ijms22105221 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5221

Author(s):

Danqi Zeng ◽

Jaime A. Teixeira da Silva ◽

Mingze Zhang ◽

Zhenming Yu ◽

Can Si ◽

...

Keyword(s):

Transcriptional Activation ◽

Flower Development ◽

Regulatory Elements ◽

Negative Control ◽

Dendrobium Officinale ◽

Luciferase Reporter ◽

Pcr Analysis ◽

Luciferase Reporter Gene ◽

Genome Wide ◽

Ap2 Transcription Factor

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

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HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0211 ◽

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.

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