Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by dual activation of the ras-phosphoinositide 3-kinase and jun n-terminal kinase pathways

Glutaredoxin 2 (Grx2) from Escherichia coli protects cerebellar neurons from dopamine-induced apoptosis via nuclear factor kappa B (NF-kappaB) activation, which is mediated by the expression of redox factor-1 (Ref-1). An analysis of the mechanisms underlying Grx2 protective activity revealed dual activation of signal transduction pathways. Grx2 significantly activated the Ras/phosphoinositide 3-kinase/Akt/NF-kappaB cascade in parallel to the Jun N-terminal kinase (JNK)/AP1 cascade. Dopamine, in comparison, down-regulated both pathways. Treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate Akt and AP-1 but had no effect on the phosphorylation of JNK1/2, suggesting that Akt/NF-kappaB and AP-1 are regulated by Ref-1. Exposure of the neurons to JNK1/2 antisense oligonucleotide in the presence of Grx2 significantly reduced AP-1 and NF-kappaB DNA binding activities and abolished Grx2 protection. These results demonstrate that dual activation of Ras/phosphoinositide 3-kinase and AP-1 cascades, which are mediated by Ref-1, is an essential component of the Grx2 mechanism of action.     

Angiotensin II and Aldosterone stimulating NF-kappaB and AP-1 activation in hepatic fibrosis of rat

BACKGROUND/AIMS: Intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis of liver. However, the signal transduction mechanism underlying effects of Angiotensin II (Ang II) and Aldosterone (Aldo) on Nuclear Factor-kappaB (NF-kappaB) and active protein-1 (AP-1) pathway in hepatic fibrogenesis remains to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying effects of Ang II and Aldo on NF-kappaB and AP-1 pathway during hepatic fibrogenesis. METHODS: To assess the effect of AECI and Angiotensin II type 1 receptor (AT-1 receptor) blocker on NF-kappaB activity in liver, a model of fibrosis was performed in rat. In vitro, hepatic stellate cells (HSCs)-T6 cells were preincubated for 1 h or not with U0126, a specific inhibitor of extracellular signal regulated kinase (ERK), irbesartan, and N-acetylcysteine prior to exposure to Ang II or Aldo for the indicated times. DNA binding activity of NF-kappaB and AP-1 were analyzed by Electrophoretic mobility shift assay (EMSA). Western blot was used to detect expression of IkappaBalpha and Phospho-P42/44. RT-PCR was used to detect the expressions of tumor necrosis factor alpha (TNFalpha) mRNA and alpha1 (I) procollagen mRNA. RESULTS: AECI and AT-1 receptor blocker exert anti-fibrosis effect through inhibiting NF-kappaB activation in liver. Ang II and Aldo increase HSCs NF-kappaB activity and NF-kappaB target gene-TNFalpha expression by inhibiting IkappaBalpha expression in a redox-sensitive manner. Ang II and Aldo also markedly increase HSCs AP-1 activity and AP-1 target gene-alpha1 (I) procollagen mRNA expression via ERK1/2 pathway in a redox-sensitive manner. CONCLUSIONS: These results show that stimulation of NF-kappaB and AP-1 pathway mediate hepatic fibrogenesis induced by intrahepatic RAAS.     

Oxygen radicals trigger activation of NF-kappaB and AP-1 and upregulation of ICAM-1 in reperfused canine heart

We investigated whether oxygen radicals generated during ischemia-reperfusion trigger postischemic inflammation in the heart. Closed-chest dogs underwent 90-min coronary artery occlusion, followed by 1- or 3-h reperfusion: 10 dogs received the cell-permeant oxygen radical scavenger N-(2-mercaptopropionyl)-glycine (MPG; 8 mg x kg(-1) x h(-1) intracoronary) beginning 5 min before reperfusion, and 9 dogs received vehicle. Blood flow (microspheres), intercellular adhesion molecule (ICAM)-1 protein expression (immunohistochemistry), ICAM-1 gene activation (Northern blotting), nuclear DNA binding activity of nuclear factor (NF)-kappaB and AP-1 (electrophoretic mobility shift assays), and neutrophil (PMN) accumulation (myeloperoxidase activity) were assessed in myocardial tissue samples. ICAM-1 protein expression was high in vascular endothelium after ischemia-reperfusion but was markedly reduced by MPG. MPG treatment also markedly decreased expression of ICAM-1 mRNA and tissue PMN accumulation. Nuclear DNA binding activities of NF-kappaB and AP-1, increased by ischemia-reperfusion, were both markedly decreased by MPG at 1 h of reperfusion. However, by 3 h, AP-1 activity was only modestly reduced by MPG and NF-kappaB activity was not significantly different from ischemic-reperfused controls. These results suggest that oxygen radicals generated in vivo during reperfusion trigger early activation of NF-kappaB and AP-1, resulting in upregulation of the ICAM-1 gene in vascular endothelium and subsequent tissue accumulation of activated PMNs.     

Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by activating NF-kappa B via Ref-1

The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites. This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis. E. coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active site, penetrates into cerebellar granule neurons and exerts its activity via NF-kappaB activation. Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity. Although DA significantly reduced NF-kappaB binding activity, Grx2 could stimulate the binding of NF-kappaB to DNA by: (i) translocating NF-kappaB from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-kappaBalpha, and (ii) activating the binding of pre existing nuclear NF-kappaB. The DNA binding activity of NF-kappaB itself was essential for neuronal survival. Overexpression of I-kappaB dominant negative gene (I-kappaB-DeltaN) in granule neurons significantly reduced their viability, irrespective of the presence of Grx2. Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate NF-kappaB binding activity. These results show that Grx2 exerts its anti apoptotic activity through the activation of Ref-1, which then activates NF-kappaB.