Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells

2003 ◽  
Vol 285 (2) ◽  
pp. F219-F229 ◽  
Author(s):  
Yves Gorin ◽  
Jill M. Ricono ◽  
Nam-Ho Kim ◽  
Basant Bhandari ◽  
Goutam Ghosh Choudhury ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Mesangial Cells ◽  
Protein Kinase B ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Ros Generation ◽  
Ang Ii ◽  
Phagocyte Nadph Oxidase

ANG II induces protein synthesis through the serine-threonine kinase Akt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) of activation of Akt/PKB particularly by G protein-coupled receptors, however, is not well characterized. We explored the role of the small GTPase Rac1, a component of the phagocyte NADPH oxidase, and the gp91 phox homologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A2 inhibition and mimicked by arachidonic acid (AA). Northern blot analysis revealed high levels of Nox4 transcript in MCs and transfection with antisense (AS) oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygen species (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well as AS Nox4 inhibited ROS generation in response to ANG II and AA, whereas constitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANG II- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition, AS Nox4 inhibited ANG II-induced protein synthesis. These data provide the first evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)H oxidase and subsequent generation of ROS mediate the effect of ANG II on Akt/PKB activation and protein synthesis in MCs.

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B

2001 ◽  
Vol 356 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Mireille CORMONT ◽  
Nadine GAUTIER ◽  
Karine ILC ◽  
Yannick Le MARCHAND-BRUSTEL
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Phosphatidylinositol 3 Kinase ◽  
Isolated Adipocytes ◽  
Phosphatidylinositol 3

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 ΔCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 ΔCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 ΔCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 ΔCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

scholarly journals The T-Cell Receptor Regulates Akt (Protein Kinase B) via a Pathway Involving Rac1 and Phosphatidylinositide 3-Kinase

2000 ◽  
Vol 20 (15) ◽  
pp. 5469-5478 ◽  
Author(s):  
Elisabeth M. Genot ◽  
Cecile Arrieumerlou ◽  
Gregory Ku ◽  
Boudewijn M. T. Burgering ◽  
Arthur Weiss ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase B ◽  
Active Form ◽  
Cell Receptor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Dominant Negative Mutant

ABSTRACT The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.

Angiotensin-(1–7)-induced activation of ERK1/2 is cAMP/protein kinase A-dependent in glomerular mesangial cells

2012 ◽  
Vol 302 (6) ◽  
pp. F784-F790 ◽  
Author(s):  
George C. Liu ◽  
Gavin Y. Oudit ◽  
Fei Fang ◽  
Joyce Zhou ◽  
James W. Scholey
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
Protein Kinase A ◽  
Egf Receptor ◽  
Mesangial Cells ◽  
Renal Physiology ◽  
Intracellular Camp ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Kinase A

The renin-angiotensin system (RAS) plays an important role in renal physiology and kidney injury. Although the cellular effects of the RAS activation are generally attributed to angiotensin II (ANG II), the recent identification of angiotensin-converting enzyme 2 has shifted the focus to other peptides including Ang-(1–7). The G protein-coupled receptor for Ang-(1–7), mas, is expressed by mesangial cells (MC) but the signal transduction pathways activated by Ang-(1–7) in MC have not been fully elucidated. Accordingly, we studied the effect of Ang-(1–7) on extracellular signal-related kinase (ERK)1/2 activation in rat MC. Ang-(1–7)-induced ERK1/2 phosphorylation in MC is time- and concentration-dependent. Pretreatment of MC with the mas receptor antagonist A-779 but not the AT1 antagonist losartan or the AT2 antagonist PD123319 abrogated ERK1/2 activation. Neither pretreatment with the NADPH oxidase inhibitors diphenyleneiodonium and apocynin nor pretreatment with the epidermal growth factor (EGF) receptor antagonists AG1478 and PD158780 attenuated Ang-(1–7)-induced activation of ERK1/2. Even though each of these compounds abolished ANG II-induced activation of ERK1/2. Ang-(1–7) increased intracellular cAMP levels and activated protein kinase A (PKA) and inhibition of either adenylyl cyclase or PKA activity attenuated Ang-(1–7)-induced ERK1/2 activation. In conclusion, Ang-(1–7)-induced activation of ERK1/2 is cAMP/PKA-dependent in MC, but independent of NADPH oxidase and the EGF receptor.

Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 1172-1180 ◽  
Author(s):  
Gwenny M. Fuhler ◽  
A. Lyndsay Drayer ◽  
Edo Vellenga
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Actin Polymerization ◽  
Protein Kinase B ◽  
Small Gtpase ◽  
Signal Transduction Pathways ◽  
Ros Generation ◽  
Ros Production ◽  
Gm Csf ◽  
Extracellular Signal

AbstractNeutrophils from patients with myelodysplastic syndrome (MDS) show a disturbed differentiation pattern and are generally dysfunctional. To study these defects in more detail, we investigated reactive-oxygen species (ROS) production and F-actin polymerization in neutrophils from MDS patients and healthy controls and the involvement of N-formyl-L-methionyl-L-lucyl-L-phenylaline (fMLP) and granulocyte macrophage–colony-stimulating factor (GM-CSF)–stimulated signal transduction pathways. Following fMLP stimulation, similar levels of respiratory burst, F-actin polymerization, and activation of the small GTPase Rac2 were demonstrated in MDS and normal neutrophils. However, GM-CSF and G-CSF priming of ROS production were significantly decreased in MDS patients. We subsequently investigated the signal transduction pathways involved in ROS generation and demonstrated that fMLP-stimulated ROS production was inhibited by the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the MAPK/ERK kinase (MEK) inhibitor U0126. In contrast, ROS production induced by fMLP stimulation of GM-CSF–primed cells was inhibited by LY294002 and U0126. This coincides with enhanced protein kinase B (PKB/Akt) phosphorylation that was PI3K dependent and enhanced extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation that was PI3K independent. We demonstrated higher protein levels of the PI3K subunit p110 in neutrophils from MDS patients and found that though the fMLP-induced phosphorylation of PKB/Akt and ERK1/2 could also be enhanced by pretreatment with GM-CSF in these patients, the degree and kinetics of PKB/Akt and ERK1/2 phosphorylation were significantly disturbed. These defects were observed despite a normal GM-CSF–induced signal transducer and activator of transcription 5 (STAT5) phosphorylation. Our results indicate that the reduced priming of neutrophil ROS production in MDS patients might be caused by a disturbed convergence of the fMLP and GM-CSF signaling routes.

Redox dependence of glomerular epithelial cell hypertrophy in response to glucose

2006 ◽  
Vol 290 (3) ◽  
pp. F741-F751 ◽  
Author(s):  
Nam-Ho Kim ◽  
Hernan Rincon-Choles ◽  
Basant Bhandari ◽  
Goutam Ghosh Choudhury ◽  
Hanna E. Abboud ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
High Glucose ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Leucine Incorporation ◽  
Ros Generation ◽  
Additive Effects ◽  
Ang Ii ◽  
Glomerular Epithelial Cell ◽  
Diphenylene Iodonium

Podocytes or glomerular epithelial cells (GECs) are important targets of the diabetic microenvironment. Podocyte foot process effacement and widening, loss of GECs and hypertrophy are pathological features of this disease. ANG II and oxidative stress are key mediators of renal hypertrophy in diabetes. The cellular mechanisms responsible for GEC hypertrophy in diabetes are incompletely characterized. We investigated the effect of high glucose on protein synthesis and GEC hypertrophy. Exposure of GECs to high glucose dose dependently stimulated [3H]leucine incorporation, but not [3H]yhymidine incorporation. High glucose resulted in the activation of ERK1/2 and Akt/PKB. ERK1/2 pathway inhibitor or the dominant negative mutant of Akt/PKB inhibited high glucose-induced protein synthesis. High glucose elicited a rapid generation of reactive oxygen species (ROS). The stimulatory effect of high glucose on ROS production, ERK1/2, and Akt/PKB activation was prevented by the antioxidants catalase, diphenylene iodonium, and N-acetylcysteine. Exposure of the cells to hydrogen peroxide mimicked the effects of high glucose. In addition, ANG II resulted in the activation of ERK1/2 and Akt/PKB and GEC hypertrophy. Moreover, high glucose and ANG II exhibited additive effects on ERK1/2 and Akt/PKB activation as well as protein synthesis. These additive responses were abolished by treatment of the cells with the antioxidants. These data demonstrate that high glucose stimulates GEC hypertrophy through a ROS-dependent activation of ERK1/2 and Akt/PKB. Enhanced ROS generation accounts for the additive effects of high glucose and ANG II, suggesting that this signaling cascade contributes to GEC injury in diabetes.

scholarly journals Evidence for a role of protein kinase C in hypoxic pulmonary vasoconstriction

1999 ◽  
Vol 276 (1) ◽  
pp. L90-L95 ◽  
Author(s):  
Norbert Weissmann ◽  
Robert Voswinckel ◽  
Thorsten Hardebusch ◽  
Simone Rosseau ◽  
Hossein Ardeschir Ghofrani ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
Superoxide Anion ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Ang Ii ◽  
Pkc Inhibitor ◽  
Pulmonary Vasoconstriction ◽  
Pressor Responses ◽  
Hypoxic Vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation, thus optimizing gas exchange. NADPH oxidase-related superoxide anion generation has been suggested as part of the signaling response to hypoxia. Because protein kinase (PK) C activation can occur during hypoxia and PKC activation is known to be critical for NADPH oxidase stimulation in different cell types, we probed the role of PKC in hypoxic vasoconstriction in intact rabbit lungs. Control vasoconstrictor responses were elicited by angiotensin II (ANG II) and the stable thromboxane analog U-46619. Portions of the experiments were performed while NO synthesis and prostanoid generation were blocked with N G-monomethyl-l-arginine and acetylsalicylic acid to avoid confounding effects due to interference with these vasoactive mediators. The PKC inhibitor H-7 (10–50 μM) caused dose-dependent inhibition of HPV, but this agent lacked specificity because ANG II- and U-46619-induced vasoconstrictions were correspondingly suppressed. In contrast, low concentrations of the specific PKC inhibitor bisindolylmaleimide I (BIM; 1–15 μM) strongly inhibited the hypoxic vasoconstriction without any interference with the responses to the pharmacological agents. Superimposable dose-inhibition curves were also obtained for BIM when lung NO synthesis and prostanoid generation were blocked throughout the experiments. Under either condition, BIM did not affect normoxic vascular tone. The PKC activator farnesylthiotriazole (FTT), ascertained to stimulate rabbit NADPH oxidase by provocation of alveolar macrophage superoxide anion generation in vitro, caused rapid-onset, transient pressor responses in normoxic lungs. After FTT, the hypoxic vasoconstrictor response was totally suppressed, in contrast to the largely maintained pressor responses to ANG II and U-46619. The lungs became refractory even to delayed hypoxic challenges after FTT application. In conclusion, these data support the concept that activation of PKC is involved in the transduction pathway forwarding pulmonary vasoconstriction in response to alveolar hypoxia.

scholarly journals PKC-α mediates flow-stimulated superoxide production in thick ascending limbs

2010 ◽  
Vol 298 (4) ◽  
pp. F885-F891 ◽  
Author(s):  
Nancy J. Hong ◽  
Guillermo B. Silva ◽  
Jeffrey L. Garvin
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Mesangial Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxidase Inhibitor ◽  
Dominant Negative ◽  
Aortic Endothelial Cells ◽  
Nadph Oxidase Activity ◽  
Measured Flow

We showed that luminal flow increases net superoxide (O2−) production via NADPH oxidase in thick ascending limbs. Protein kinase C (PKC) activates NADPH oxidase activity in phagocytes, cardiomyocytes, aortic endothelial cells, vascular smooth muscle cells, and renal mesangial cells. However, the flow-activated pathway that induces NADPH oxidase activity in thick ascending limbs is unclear. We hypothesized that PKC mediates flow-stimulated net O2− production by thick ascending limbs. Initiation of flow (20 nl/min) increased net O2− production from 4 ± 1 to 61 ± 12 AU/s ( P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net O2− production (2 ± 1 vs. 1 ± 1 AU/s; P > 0.05; n = 5). Flow-stimulated O2− was also blocked in p47phox-deficient mice. We measured flow-stimulated PKC activity with a fluorescence resonance energy transfer (FRET)-based membrane-targeted PKC activity reporter and found that the FRET ratio increased from 0.87 ± 0.02 to 0.96 ± 0.04 AU ( P < 0.05; n = 6). In the absence of flow, the PKC activator phorbol 12-myristate 13-acetate (200 nM) enhanced net O2− production from 5 ± 2 to 92 ± 6 AU/s ( P < 0.001; n = 6). The PKC-α- and βI-selective inhibitor Gö 6976 (100 nM) decreased flow-stimulated net O2− production from 54 ± 15 to 2 ± 1 AU/s ( P < 0.04; n = 5). Flow-induced net O2− production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-α but not dnPKCβI or LacZ (Δ = 11 ± 3 AU/s for dnPKCα, 55 ± 7 AU/s for dnPKCβI, and 63 ± 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net O2− production in thick ascending limbs via PKC-α-mediated activation of NADPH oxidase.

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