Activation of PI3K-Akt pathway mediates antiapoptotic effects of β-adrenergic agonist in airway eosinophils

2005 ◽  
Vol 288 (5) ◽  
pp. L860-L867 ◽  
Author(s):  
Kentaro Machida ◽  
Hiromasa Inoue ◽  
Koichiro Matsumoto ◽  
Miyuki Tsuda ◽  
Satoru Fukuyama ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Survival Signal ◽  
Eosinophil Survival ◽  
Mitogen Activated Protein ◽  
Allergen Provocation ◽  
Eosinophil Apoptosis

β-Adrenoceptor agonists reportedly decrease spontaneous apoptosis of peripheral blood eosinophils; however, its signaling pathway is unknown. Survival signals can be elicited by the activation of phosphatidylinositol 3-kinase (PI3K) and Akt, both of which are known to be potent regulators of apoptosis, and Akt in turn inactivates Forkhead transcription factors, including FKHR (Forkhead in rhabdomyosarcoma). We have investigated the effect of β-agonists on apoptosis of local eosinophils isolated from the airways and the involvement of PI3K, Akt, and FKHR in its survival signal. Eosinophils obtained from immunized mice by bronchoalveolar lavage after allergen provocation underwent apoptosis in a time-dependent manner. Incubation of eosinophils with isoproterenol or formoterol dose-dependently inhibited both spontaneous eosinophil apoptosis and apoptosis induced by Fas receptor activation. Incubation with cAMP or forskolin also inhibited eosinophil apoptosis. The PI3K inhibitors wortmannin and LY-294002 and an Akt inhibitor, 1-l-6-hydroxymethyl- chiro-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate, but not a mitogen-activated protein kinase kinase inhibitor PD-98059, blocked isoproterenol-mediated eosinophil survival. Wortmannin also inhibited cAMP-mediated eosinophil survival. Isoproterenol rapidly induced phosphorylation of Akt and FKHR in eosinophils in a PI3K-dependent manner. These findings indicate that the PI3K-Akt-FKHR pathway conveys a critical survival signal induced by β-agonists in airway eosinophils.

Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes

2002 ◽  
Vol 282 (6) ◽  
pp. L1324-L1329 ◽  
Author(s):  
Andre Kulisz ◽  
Ningfang Chen ◽  
Navdeep S. Chandel ◽  
Zuohui Shao ◽  
Paul T. Schumacker
Keyword(s):  
Kinase Inhibitor ◽  
Anion Channel ◽  
P38 Map Kinase ◽  
Proximal Region ◽  
Mitogen Activated Protein Kinase ◽  
Ros Generation ◽  
Dependent Manner ◽  
Adaptive Responses ◽  
Mitochondrial Ros ◽  
Mitogen Activated Protein

The p38 mitogen-activated protein kinase (MAPK) is phosphorylated in response to oxidative stress. Mitochondria in cardiomyocytes increase their generation of reactive oxygen species (ROS) during hypoxia (1–5% O2). These ROS participate in signal transduction pathways involved in adaptive responses, including ischemic preconditioning and gene transcription. The present study therefore tested the hypothesis that hypoxia induces p38 MAPK phosphorylation by augmenting mitochondrial ROS generation. In cardiomyocytes, phosphorylation of p38 was observed in a Po 2-dependent manner during hypoxia. This response was inhibited by rotenone, thenoyltrifluoroacetone, and myxothiazol, inhibitors of mitochondrial complexes I, II, and III, respectively. A similar inhibition was observed in the cells pretreated with anion channel inhibitor DIDS, which may block ROS release from mitochondria. During normoxia, increases in mitochondrial ROS elicited by azide (1–2 mM) or by the mitochondrial inhibitor antimycin A caused increased phosphorylation of p38. Brief treatment with exogenous H2O2 during normoxia also induced phosphorylation of p38 as hypoxia, but this effect was not abolished by myxothiazol or DIDS. The antioxidant N-acetyl-cysteine abolished the p38 response to hypoxia, presumably by scavenging H2O2, but the mitogen extracellular receptor kinase inhibitor PD-98059 did not inhibit p38 phosphorylation during hypoxia. Thus physiological hypoxia leads to p38 phosphorylation through a mechanism that requires electron flux in the proximal region of the mitochondrial electron transport chain, which suggests that either H2O2 or superoxide participates in activating that process.

CCK activates p90rsk in rat pancreatic acini through protein kinase C

1997 ◽  
Vol 272 (3) ◽  
pp. G401-G407 ◽  
Author(s):  
M. J. Bragado ◽  
A. Dabrowski ◽  
G. E. Groblewski ◽  
J. A. Williams
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Threshold Concentration ◽  
Mitogen Activated Protein Kinase ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Mitogen Activated Protein

The presence of the 90-kDa ribosomal S6 protein kinase (p90(rsk)) in isolated rat pancreatic acini was demonstrated by Western blotting and immunoprecipitation with anti-p90(rsk). Cholecystokinin (CCK) activated p90(rsk) activity in a time- and dose-dependent manner and increased its phosphorylation. The threshold concentration of CCK was 10 pM and the maximal effect was seen at 1 nM. An increase in p90(rsk) was observed 1 min after 1 nM CCK stimulation, reaching a maximum at 10 min, when p90(rsk) activity was increased 5.4-fold. Carbachol and bombesin, but not vasoactive intestinal peptide, also activated p90(rsk). CCK-induced activation of p90(rsk) appears to be mediated by protein kinase C (PKC), since 12-O-tetradecanoylphorbol-13-acetate increased p90(rsk) activity 5.3-fold. GF-109293X, a potent inhibitor of PKC, strongly inhibited CCK-evoked p90(rsk) activity. Treatment of acini with ionomycin or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no effect, indicating that mobilization of intracellular Ca2+ by CCK is not important in p90(rsk) activation. Although there were some quantitative differences in the extent of inhibition, the specific inhibitors [rapamycin, wortmannin, mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059, and GF-109293X] had parallel effects on p90(rsk) and p42(mapk) activities, consistent with a model in which p90(rsk) can be regulated in acini by MAPK.

scholarly journals Mitogen-Activated Protein Kinase-Dependent Stimulation of Proliferation of Rat Lactotrophs in Culture by 3′,5′-Cyclic Adenosine Monophosphate*

Endocrinology ◽  
1999 ◽  
Vol 140 (6) ◽  
pp. 2850-2858 ◽  
Author(s):  
Shinichi Suzuki ◽  
Isao Yamamoto ◽  
Jun Arita
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Dopamine Receptor Agonist ◽  
Intracellular Camp ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Stimulatory Action ◽  
Mitogen Activated Protein ◽  
Brdu Labeling

Abstract Intracellular cAMP regulates cell proliferation as a second messenger of extracellular signals in a number of cell types. We investigated, by pharmacological means, whether an increase in intracellular cAMP levels changes proliferation rates of lactotrophs in primary culture, whether there are interactions between signal transduction pathways of cAMP and the growth factor insulin, and where the dopamine receptor agonist bromocriptine acts in the cAMP pathway to inhibit lactotroph proliferation. Rat anterior pituitary cells, cultured in serum-free medium, were treated with cAMP-increasing agents, followed by 5-bromo-2′-deoxyuridine (BrdU) to label proliferating pituitary cells. BrdU-labeling indices indicative of the proliferation rate of lactotrophs were determined by double immunofluorescence staining for PRL and BrdU. Treatment with forskolin (an adenylate cyclase activator) or (Bu)2cAMP (a membrane-permeable cAMP analog) increased BrdU-labeling indices of lactotrophs in a dose- and incubation time-dependent manner. The cAMP-increasing agents were also effective in increasing BrdU-labeling indices in populations enriched for lactotrophs by differential sedimentation. The stimulatory action of forskolin was observed, regardless of concentrations of insulin that were added in combination with forskolin. Inhibition of the action of endogenous cAMP by H89 or KT5720, a protein kinase A inhibitor, attenuated an increase in BrdU-labeling indices by insulin treatment. On the other hand, the specific mitogen-activated protein kinase inhibitor PD98059, which was effective in blocking the mitogenic action of insulin, markedly suppressed the forskolin-induced increase in BrdU-labeling indices. (Bu)2cAMP antagonized not only inhibition of BrdU labeling indices but also changes in cell shape induced by bromocriptine treatment, although forskolin did not have such an antagonizing effect. These results suggest that: 1) intracellular cAMP plays a stimulatory role in the regulation of lactotroph proliferation; 2) cAMP and insulin/mitogen-activated protein kinase signalings require each other for their mitogenic actions; and 3) the antimitogenic action of bromocriptine is, at least in part, caused by inhibition of cAMP production.

Role of tyrosine kinase and p44/42 MAPK in D2-like receptor-mediated stimulation of Na+, K+-ATPase in kidney

2002 ◽  
Vol 282 (4) ◽  
pp. F697-F702 ◽  
Author(s):  
Vihang Narkar ◽  
Tahir Hussain ◽  
Mustafa Lokhandwala
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Rat Kidney ◽  
Receptor Activation ◽  
Proximal Tubules ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Our laboratory has shown that dopamine D2-like receptor activation causes stimulation of Na+, K+-ATPase (NKA) activity in the proximal tubules of the rat kidney. The present study was designed to investigate the cellular signaling mechanisms mediating this response to D2-like receptor activation. We measured the stimulation of NKA activity by bromocriptine (D2-like receptor agonist) in the absence and presence of PD-98059 [p44/42 mitogen-activated protein kinase (MAPK) kinase inhibitor] and genistein (tyrosine kinase inhibitor) in renal proximal tubules. Both agents inhibited bromocriptine-mediated stimulation of NKA, suggesting the involvement of p44/42 MAPK and tyrosine kinase in this response. Additionally, we found that bromocriptine increased the phosphorylation of p44/42 MAPK in the proximal tubules, which was blocked by PD-98059 and genistein. These results show that D2-like receptor activation causes stimulation of NKA activity by means of a tyrosine kinase-p44/42 MAPK pathway in the proximal tubules of the kidney.

scholarly journals Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3410-3414 ◽  
Author(s):  
Analia Garcia ◽  
Todd M. Quinton ◽  
Robert T. Dorsam ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Kinase Inhibitor ◽  
Thromboxane A2 ◽  
Receptor Activation ◽  
Src Family Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Src Family Kinase

AbstractThe binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non–aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.

Human Caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner

2000 ◽  
Vol 278 (6) ◽  
pp. G952-G966 ◽  
Author(s):  
Cheng Fang Yu ◽  
Matthew A. Sanders ◽  
Marc D. Basson
Keyword(s):  
Focal Adhesion ◽  
Kinase Inhibitor ◽  
Mucosal Healing ◽  
Matrix Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
P38 Inhibitor ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

The signals involved in restitution during mucosal healing are poorly understood. We compared focal adhesion kinase (FAK) and paxillin protein and phosphorylation, extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 activation, as well as FAK and paxillin organization in static and migrating human intestinal Caco-2 cells on matrix proteins and anionically derivatized polystyrene dishes (tissue culture plastic). We also studied effects of FAK, ERK, and p38 blockade in a monolayer-wounding model. Compared with static cells, cells migrating across matrix proteins matrix-dependently decreased membrane/cytoskeletal FAK and paxillin and cytosolic FAK. Tyrosine phosphorylated FAK and paxillin changed proportionately to FAK and paxillin protein. Conversely, cells migrating on plastic increased FAK and paxillin protein and phosphorylation. Migration matrix-dependently activated p38 and inactivated ERK1 and ERK2. Total p38, ERK1, and ERK2 did not change. Caco-2 motility was inhibited by transfection of FRNK (the COOH-terminal region of FAK) and PD-98059, a mitogen-activated protein kinase-ERK kinase inhibitor, but not by SB-203580, a p38 inhibitor, suggesting that FAK and ERK modulate Caco-2 migration. In contrast to adhesion-induced phosphorylation, matrix may regulate motile intestinal epithelial cells by altering amounts and distribution of focal adhesion plaque proteins available for phosphorylation as well as by p38 activation and ERK inactivation. Motility across plastic differs from migration across matrix.

scholarly journals Mechanically activated Piezo1 channels of cardiac fibroblasts stimulate p38 mitogen-activated protein kinase activity and interleukin-6 secretion

2019 ◽  
Vol 294 (46) ◽  
pp. 17395-17408 ◽  
Author(s):  
Nicola M. Blythe ◽  
Katsuhiko Muraki ◽  
Melanie J. Ludlow ◽  
Vasili Stylianidis ◽  
Hamish T. J. Gilbert ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Cardiac Fibroblasts ◽  
Ruthenium Red ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Mitogen Activated Protein

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/− cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.

scholarly journals Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of endothelial prostacyclin production

1996 ◽  
Vol 320 (1) ◽  
pp. 221-226 ◽  
Author(s):  
Viral PATEL ◽  
Colin BROWN ◽  
Adele GOODWIN ◽  
Neil WILKIE ◽  
Michael R BOARDER
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Western Blots ◽  
Mitogen Activated Protein ◽  
Two Peaks ◽  
Stimulation Of

Extracellular ATP and ADP, released from platelets and other sites stimulate the endothelial production of prostacyclin (PGI2) by acting on G-protein-coupled P2Y1 and P2Y2 purinoceptors, contributing to the maintenance of a non-thrombogenic surface. The mechanism, widely described as being dependent on elevated cytosolic [Ca2+], also requires protein tyrosine phosphorylation. Here we show that activation of both these P2 receptor types leads to the tyrosine phosphorylation and activation of both the p42 and p44 forms of mitogen-activated protein kinase (MAPK). 2-Methylthio-ATP and UTP, selectively activating P2Y1 and P2Y2 purinoceptors respectively, and ATP, a non-selective agonist at these two receptors, stimulate the tyrosine phosphorylation of both p42mapk and p44mapk, as revealed by Western blots with an antiserum specific for the tyrosine-phosphorylated forms of the enzymes. By using separation on Resource Q columns, peptide kinase activity associated with the phosphorylated MAPK enzymes distributes into two peaks, one mainly p42mapk and one mainly p44mapk, both of which are stimulated by ATP with respect to kinase activity and phospho-MAPK immunoreactivity. Stimulation of P2Y1 or P2Y2 purinoceptors leads to a severalfold increase in PGI2 efflux; this was blocked in a dose-dependent manner by the selective MAPK kinase inhibitor PD98059. This drug also blocked the agonist-stimulated increase in phospho-MAPK immunoreactivity for both p42mapk and p44mapk but left the phospholipase C response to P2 agonists essentially unchanged. Olomoucine has been reported to inhibit p44mapk activity. Here we show that in the same concentration range olomoucine inhibits activity in both peaks from the Resource Q column and also the agonist stimulation of 6-keto-PGF1, but has no effect on agonist-stimulated phospho-MAPK immunoreactivity. These results provide direct evidence for the involvement of p42 and p44 MAPK in the PGI2 response of intact endothelial cells: we have shown that both the endothelial P2Y purinoceptors are linked to activation of MAPK, and that activation of this pathway is a requirement for the stimulation by ATP/ADP of endothelial PGI2 production.

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