scholarly journals Novel protein kinase C-epsilon inhibits human CYP11B2 gene expression through ERK1/2 signalling pathway and JunB

2006 ◽  
Vol 36 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Jean-Guy LeHoux ◽  
Andrée Lefebvre
Keyword(s):  
Protein Kinase ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Ang Ii ◽  
Protein Levels ◽  
Protein Kinase C Epsilon ◽  
Dependent Protein ◽  
H295r Cells

We previously reported that H295R cells co-express three diacylglycerol (DAG)-dependent protein kinase Cs (PKCs), namely conventional (c) PKCα and novel (n) PKCε and PKCϑ. The aim of the present work was to evaluate the implication of DAG-dependent PKCs in the activation of p44/42 MAP kinase (MAPK) by angiotensin II (Ang II) and to define the role of this pathway towards CYP11B2 regulation in H295R cells. The PKC inhibitor bisindolylmaleimide 1 (Bis) inhibited Ang II-induced p44/42 MAPK phosphorylation whereas the cPKC inhibitor Gö6976 failed to do so, thus ruling out the participation of PKCα. Ang II activated nPKCε and did not affect nPKCϑ, pinpointing PKCε as the mediator of Ang II in p44/42 MAPK activation. Overexpression of wild-type ERK1 and ERK2 significantly reduced basal as well as Ang II-stimulated human -2023CYP11B2-CAT activity; conversely, the two dominant negative mutants increased them. Overexpression of constitutively active (ca) PKCsuppressed Ang II-induced -2023CYP11B2-CAT activity. Infection of H295R cells with adenoviruses (Adv) expressing caPKCε activated endogenous MEK1/2 and p44/42 MAPK. Adv-caPKCε inhibited Ang II-stimulated aldosterone synthase mRNA levels and this action was reversed by the MEK1 inhibitor, PD98059. Also, Ang II increased JunB protein levels and this effect was inhibited by PD98059 and Bis. Adv-caPKCε enhanced JunB protein levels and PD98059 attenuated the increase. JunB overexpression abolished the Ang II-induced promoter activity within -138 bp of the 5′-flanking region of CYP11B2. Collectively, these results demonstrate that PKCε inhibits CYP11B2 transcription through the p44/42 MAPK pathway and JunB in H295R cells.

scholarly journals Double-stranded RNA-dependent protein kinase activation modulates endotoxin-induced diaphragm weakness

2011 ◽  
Vol 110 (1) ◽  
pp. 199-205 ◽  
Author(s):  
G. S. Supinski ◽  
L. A. Callahan
Keyword(s):  
Protein Kinase ◽  
Time Course ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Kinase Activation ◽  
Protein Levels ◽  
Dependent Protein ◽  
Diaphragm Weakness

Diaphragm caspase-8 activation plays a key role in modulating sepsis-induced respiratory muscle dysfunction. It is also known that double-stranded RNA-dependent protein kinase (PKR) is a regulator of caspase-8 activation in neural tissue. We tested the hypothesis that the PKR pathway modulates sepsis-induced diaphragmatic caspase-8 activation. We first evaluated the time course of diaphragm PKR activation following endotoxin administration in mice. We then determined whether administration of a PKR inhibitor (2-aminopurine) prevents endotoxin-induced diaphragm caspase-8 activation and contractile dysfunction in mice. Finally, we investigated if inhibition of PKR (using either 2-aminopurine or transfection with dominant-negative PKR) blocks caspase-8 activation in cytokine treated C2C12 cells. Endotoxin markedly activated diaphragm PKR (with increases in both active phospho-PKR protein levels, P < 0.03, and directly measured PKR activity, P < 0.01) and increased active caspase-8 levels ( P < 0.01). Inhibition of PKR with 2-aminopurine prevented endotoxin-induced diaphragm caspase-8 activation ( P < 0.01) and diaphragm weakness ( P < 0.001). Inhibition of PKR with either 2-aminopurine or transfection with dominant-negative PKR blocked caspase-8 activation in isolated cytokine-treated C2C12 cells. These data implicate PKR activation as a major factor mediating cytokine-induced skeletal muscle caspase-8 activation and weakness.

Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis

2005 ◽  
Vol 289 (5) ◽  
pp. E857-E863 ◽  
Author(s):  
Anastasia Kariagina ◽  
Svetlana Zonis ◽  
Mahta Afkhami ◽  
Dmitry Romanenko ◽  
Vera Chesnokova
Keyword(s):  
Glucocorticoid Receptor ◽  
Hpa Axis ◽  
Leukemia Inhibitory Factor ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Inhibitory Factor ◽  
Mrna Levels ◽  
Hypothalamic Pituitary Adrenal ◽  
Protein Levels

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the gp130 family. LIF is induced peripherally and within the brain during inflammatory or chronic autoimmune diseases and is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here we investigated the role of LIF in mediating glucocorticoid receptor (GR) expression in the HPA axis. LIF treatment (3 μg/mouse, ip) markedly decreased GR mRNA levels in murine hypothalamus (5-fold, P < 0.01) and pituitary (1.7-fold, P < 0.01) and downregulated GR protein levels. LIF decreased GR expression in murine corticotroph cell line AtT20 within 2 h, and this effect was sustained for 8 h after treatment. LIF-induced GR mRNA reduction was abrogated in AtT20 cells overexpressing dominant-negative mutants of STAT3, indicating that intact JAK-STAT signaling is required to mediate LIF effects on GR expression. Conversely, mice with LIF deficiency exhibited increased GR mRNA levels in the hypothalamus and pituitary (3.5- and 3.5-fold, respectively; P < 0.01 for both) and increased GR protein expression when compared with wild-type littermates. The suppressive effects of dexamethasone on GR were more pronounced in LIF-null animals. These data suggest that LIF maintains the HPA axis activation by decreasing GR expression and raise the possibility that LIF might contribute to the development of central glucocorticoid resistance during inflammation.

scholarly journals Mechanisms of angiotensin II-induced expression of B2 kinin receptors

2004 ◽  
Vol 286 (3) ◽  
pp. H926-H932 ◽  
Author(s):  
Yan Tan ◽  
Florence N. Hutchison ◽  
Ayad A. Jaffa
Keyword(s):  
Receptor Antagonist ◽  
Mapk Pathway ◽  
Renin Angiotensin System ◽  
Receptor Subtype ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Ang Ii ◽  
Western Blots ◽  
Protein Levels ◽  
Kinin Receptors

Although the primary roles of the kallikreinkinin system and the renin-angiotensin system are quite divergent, they are often intertwined under pathophysiological conditions. We examined the effect of ANG II on regulation of B2 kinin receptors (B2KR) in vascular cells. Vascular smooth muscle cells (VSMC) were treated with ANG II in a concentration (10—9-10—6 M)- and time (0–24 h)-dependent manner, and B2KR protein and mRNA levels were measured by Western blots and PCR, respectively. A threefold increase in B2KR protein levels was observed as early as 6 h, with a peak response at 10—7 M. ANG II (10—7 M) also increased B2KR mRNA levels twofold 4 h after stimulation. Actinomycin D suppressed the increase in B2KR mRNA and protein levels induced by ANG II. To elucidate the receptor subtype involved in mediating this regulation, VSMC were pretreated with losartan (AT1 receptor antagonist) and/or PD-123319 (AT2 receptor antagonist) at 10 μM for 30 min, followed by ANG II (10—7 M) stimulation. Losartan completely blocked the ANG II-induced B2KR increase, whereas PD-123319 had no effect. In addition, expression of B2KR mRNA levels was decreased in AT1A receptor knockout mice. Finally, to determine whether ANG II stimulates B2KR expression via activation of the MAPK pathway, VSMC were pretreated with an inhibitor of p42/p44mapk (PD-98059) and/or an inhibitor of p38mapk (SB-202190), followed by ANG II (10—7 M) for 24 h. Selective inhibition of the p42/p44mapk pathway significantly blocked the ANG II-induced increase in B2KR expression. These findings demonstrate that ANG II regulates expression of B2KR in VSMC and provide a rationale for studying the interaction between ANG II and bradykinin in the pathogenesis of vascular dysfunction.

scholarly journals BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites

2006 ◽  
Vol 26 (24) ◽  
pp. 9497-9507 ◽  
Author(s):  
Don Benjamin ◽  
Martin Schmidlin ◽  
Lu Min ◽  
Brigitte Gross ◽  
Christoph Moroni
Keyword(s):  
Protein Kinase ◽  
Mrna Decay ◽  
Protein Kinase B ◽  
Proteasomal Degradation ◽  
Mrna Levels ◽  
Cell Compartment ◽  
Protein Levels ◽  
Dependent Protein

ABSTRACT BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBα−/− cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

Role of AT1 and AT2 receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes

1998 ◽  
Vol 275 (3) ◽  
pp. H906-H916 ◽  
Author(s):  
Thomas A. Fischer ◽  
Krishna Singh ◽  
Donald S. O’Hara ◽  
David M. Kaye ◽  
Ralph A. Kelly
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Ventricular Myocytes ◽  
Mrna Levels ◽  
Ang Ii ◽  
Terminal Protein ◽  
Erk Activity ◽  
Angiotensin Type

ANG II has been implicated in the hypertrophic response in ventricular myocytes by acting at the angiotensin type 1 (AT1) receptor. However, the role of the angiotensin type 2 (AT2) receptor in the adult heart is not as clearly understood. In adult rat ventricular myocytes (ARVM) and cardiac microvascular endothelial cells (CMEC), we examined the role of ANG II signaling, via AT1 and AT2 receptors, on the activation of the extracellular signal-regulated protein kinases (ERKs) and on the expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1. ANG II caused no detectable increase in ERK activity or in c- fos mRNA abundance in ARVM but increased ERK activity within 5 min in CMEC and increased c- fos mRNA levels. However, in the presence of the selective phosphoprotein phosphatase (PP-2A/PP-1) inhibitor okadaic acid (OA), a sustained increase in ERK activity, as well as in c- junNH2-terminal protein kinase activity, in ARVM was observed. ANG II increased MKP-1 mRNA levels within 15 min in ARVM and CMEC. In contrast to the response in endothelial cells, however, ANG II activation of MKP-1 in ARVM was mediated by AT2-receptor activation. Thus there is constitutive as well as inducible suppression of ERKs and c- junNH2-terminal protein kinases by MKP and PP-2A/PP-1 in the adult cardiac myocyte phenotype.

Angiotensin II Decreases Neuronal Delayed Rectifier Potassium Current: Role of Calcium/Calmodulin-Dependent Protein Kinase II

1999 ◽  
Vol 82 (3) ◽  
pp. 1560-1568 ◽  
Author(s):  
Mingyan Zhu ◽  
Craig H. Gelband ◽  
Philip Posner ◽  
Colin Sumners
Keyword(s):  
Angiotensin Ii ◽  
Protein Kinase ◽  
Brain Stem ◽  
Delayed Rectifier ◽  
Ang Ii ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Mediated Reduction

Angiotensin II (Ang II) acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion. These actions of Ang II are mediated via Ang II type 1 (AT1) receptors and involve modulation of membrane ionic currents and neuronal activity. In previous studies we utilized neurons cultured from the hypothalamus and brain stem of newborn rats to investigate the AT1receptor–mediated effects of Ang II on neuronal K+currents. Our data indicate that Ang II decreases neuronal delayed rectifier (Kv) current, and that this effect is partially due to activation of protein kinase C (PKC), specifically PKCα. However, the data also indicated that another Ca2+-dependent mechanism was also involved in addition to PKC. Because Ca2+/calmodulin-dependent protein kinase II (CaM KII) is a known modulator of K+ currents in neurons, we investigated the role of this enzyme in the AT1 receptor–mediated reduction of neuronal Kv current by Ang II. The reduction of neuronal Kv current by Ang II was attenuated by selective inhibition of either calmodulin or CaM KII and was mimicked by intracellular application of activated (autothiophosphorylated) CaM KIIα. Concurrent inhibition of CaM KII and PKC completely abolished the reduction of neuronal Kv by Ang II. Consistent with these findings is the demonstration that Ang II increases CaM KII activity in neuronal cultures, as evidenced by increased levels of autophosphorylated CaM KIIα subunit. Last, single-cell reverse transcriptase (RT)-PCR analysis revealed the presence of AT1 receptor-, CaM KIIα-, and PKCα subunit mRNAs in neurons that responded to Ang II with a decrease in Kv current. The present data indicate that the AT1receptor–mediated reduction of neuronal Kv current by Ang II involves a Ca2+/calmodulin/CaM KII pathway, in addition to the previously documented involvement of PKC.

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