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

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.

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A Specific Role of Phosphatidylinositol 3–Kinase γ

The Journal of Cell Biology ◽

10.1083/jcb.152.4.717 ◽

2001 ◽

Vol 152 (4) ◽

pp. 717-728 ◽

Cited By ~ 39

Author(s):

Claire Bony ◽

Serge Roche ◽

Ueno Shuichi ◽

Takehiko Sasaki ◽

Michael A. Crackower ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cardiac Cells ◽

Dominant Negative ◽

Neonatal Rat ◽

Dominant Negative Mutant ◽

Cardiac Cell ◽

Autonomic Activity ◽

Cell Functions ◽

Negative Mutant ◽

Stimulation Of

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase–dependent pathway that stimulates PLCγ and generates IP3, a breakdown product of phosphatidylinositol 4,5–bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca2+ spiking). PIP2 is phosphorylated on 3′ by phosphoinositide 3–kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5–trisphosphate, regulates activity of PLCγ. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCγ, Ca2+ homeostasis. However, although PI3Kα and -β have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kγ. We report that neonatal rat cardiac cells in culture express PI3Kα, -β, and -γ. The purinergic agonist predominantly activates PI3Kγ. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCγ as well as IP3 generation in ATP-stimulated cells. Furthermore, an anti-PI3Kγ, but not an anti-PI3Kβ, injected in the cells prevents the effect of ATP on cell Ca2+ spiking. A dominant negative mutant of PI3Kγ transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca2+ spiking of wild-type but not of PI3Kγ2/2 embryonic stem cell–derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCγ. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca2+ spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kγ antibody and a dominant negative mutant of PI3Kγ injected or transfected into cells prevent the latter event. We conclude that PI3Kγ activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca2+ spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kγ to fully activate PLCγ in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP3 generation and thus cardiac autonomic activity.

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Abstract 11985: C1q / tnf-Related Protein 9 Protects Against Acute Myocardial Injury

Circulation ◽

10.1161/circ.130.suppl_2.11985 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Takahiro Kambara ◽

Rei Shibata ◽

Koji Ohashi ◽

Yuusuke Joki ◽

Satoko Hayakawa ◽

...

Keyword(s):

Cardiac Myocytes ◽

Inflammatory Cytokines ◽

Ischemia Reperfusion ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Mutant Form ◽

Related Protein ◽

Increased Risk ◽

Tnf Α ◽

Negative Mutant

Background: Obesity-related disorders are associated with an increased risk for cardiovascular disease. C1q/TNF-related protein (CTRP) 9 is an anti-diabetic adipokine that is downregulated in obese mice. Recently, we have reported that CTRP9 prevents adverse vascular remodeling in response to injury. Here, we investigated the effect of CTRP9 on acute cardiac injury with loss-of-function genetic manipulations. Methods and Results: CTRP9-deficient (CTRP9-KO) mice at the age of 12 weeks were indistinguishable from Wild-type (WT) mice under basal conditions. CTRP9-KO mice showed increased myocardial infarct size and elevated expression of inflammatory cytokines including TNF-α and IL-6 in ischemic heart following ischemia-reperfusion compared with WT mice. CTRP9-KO mice also had exacerbated contractile left ventricle (LV) dysfunction and increased myocardial expression of inflammatory cytokines following intraperitoneal injection of lipopolysaccharide (LPS) compared with WT mice. Conversely, systemic delivery of an adenoviral vector expressing CTRP9 (Ad-CTRP9) attenuated inflammatory responses to myocardial ischemia-reperfusion or LPS administration in WT mice. In cultured cardiac myocytes, treatment with CTRP9 protein suppressed LPS-stimulated expression of TNF-α and IL-6 with an accompanying reduction of NFkB phosphorylation. Treatment of cardiac myocytes with CTRP9 enhanced AMPK phosphorylation, and transduction with dominant-negative mutant form of AMPK reversed the suppressive effect of CTRP9 on TNF-α and IL-6 expression. Furthermore, systemic administration of Ad-CTRP9 improved LPS-induced cardiac dysfunction in WT mice but not in muscle-specific transgenic mice expressing dominant-negative mutant form of AMPK. Conclusions: These findings suggest that CTRP9 protects against acute cardiac damage in response to pathological stimuli by suppressing inflammatory reactions through AMPK-dependent mechanisms.

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Transcriptional Response to Calcium-Sensing Receptor Stimulation

Endocrinology ◽

10.1210/en.2012-1343 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4716-4728 ◽

Cited By ~ 35

Author(s):

Gerald Thiel ◽

Andrea Lesch ◽

Anja Keim

Keyword(s):

Gene Transcription ◽

Ternary Complex ◽

Dominant Negative ◽

Signaling Cascade ◽

Dominant Negative Mutant ◽

Calcium Sensing Receptor ◽

Receptor Stimulation ◽

Calcium Sensing ◽

Negative Mutant ◽

Stimulation Of

Abstract Elevated extracellular Ca2+ concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. Here we show that this stimulation induces the expression of biologically active early growth response protein 1 (Egr-1), a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of calcium-sensing receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that stimulation of calcium-sensing receptors increased the transcriptional activation potential of Elk-1. In addition, activator protein-1 (AP-1) transcriptional activity was significantly elevated after the stimulation of calcium-sensing receptors. The expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression and prevented the up-regulation of AP-1 activity as a result of calcium-sensing receptor stimulation, indicating that ternary complex factors control both Egr-1- and AP-1-regulated transcription. In addition, AP-1 activity was reduced after the expression of a dominant-negative mutant of c-Jun in cells expressing an activated calcium-sensing receptor. Stimulus-transcription coupling leading to the up-regulation of Egr-1 and AP-1 controlled transcription in cells expressing calcium-sensing receptors required the protein kinases Raf and ERK, whereas the overexpression of MAPK phosphatase-1 interrupted the signaling cascade connecting calcium-sensing receptor stimulation with transcription of Egr-1 and AP-1 controlled genes. The fact that calcium-sensing receptor stimulation activates the transcription factors Egr-1, Elk-1, and AP-1 indicates that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling.

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Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-κB in Endothelial Cells

Infection and Immunity ◽

10.1128/iai.71.8.4414-4420.2003 ◽

2003 ◽

Vol 71 (8) ◽

pp. 4414-4420 ◽

Cited By ~ 107

Author(s):

Xianwu Li ◽

Joan C. Tupper ◽

Douglas D. Bannerman ◽

Robert K. Winn ◽

Christopher J. Rhodes ◽

...

Keyword(s):

Endothelial Cells ◽

Kinase Activity ◽

Luciferase Activity ◽

Pi3 Kinase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Toll Like Receptor 4 ◽

Kinase Activation ◽

Phosphoinositide 3 Kinase ◽

Negative Mutant

ABSTRACT Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-κB and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-κB activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1β. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1β-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-κB luciferase activity, IL-6 production, and IκBα degradation elicited by LPS and IL-1β but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-κB luciferase activity evoked by LPS and IL-1β. However, expression of a constitutively activated Akt failed to induce NF-κB luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1β signaling leading to NF-κB activation in endothelial cells and that Akt is necessary but not sufficient for NF-κB activation by TLR4.

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Pathophysiological Remodeling of Mouse Cardiac Myocytes Expressing Dominant Negative Mutant of Neuron Restrictive Silencing Factor

Circulation Journal ◽

10.1253/circj.cj-10-0652 ◽

2010 ◽

Vol 74 (12) ◽

pp. 2712-2719 ◽

Cited By ~ 11

Author(s):

Makoto Takano ◽

Hideyuki Kinoshita ◽

Takao Shioya ◽

Masayuki Itoh ◽

Kazuwa Nakao ◽

...

Keyword(s):

Cardiac Myocytes ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Negative Mutant

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TNF-α activates protein synthesis in neonatal rat ventricular cardiomyocytes through NF-κB and PI3-kinase

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2008.02.257 ◽

2008 ◽

Vol 44 (4) ◽

pp. 819

Author(s):

Isabelle Plaisance ◽

Christian Morandi ◽

Marijke Brink

Keyword(s):

Protein Synthesis ◽

Pi3 Kinase ◽

Neonatal Rat ◽

Ventricular Cardiomyocytes ◽

Tnf Α

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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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