c-Jun NH2-terminal Protein Kinase Research Articles

The Cytoprotective Effect of N-acetyl-L-cysteine against ROS-Induced Cytotoxicity Is Independent of Its Ability to Enhance Glutathione Synthesis

2,3,5-Tris(glutathion-S-yl)-hydroquinone (TGHQ), a metabolite of hydroquinone, is toxic to renal proximal tubule epithelial cells. TGHQ retains the ability to redox cycle and create an oxidative stress. To assist in elucidating the contribution of reactive oxygen species (ROS) to TGHQ-induced toxicity, we determined whether the antioxidant, N-acetyl-L-cysteine (NAC), could protect human kidney proximal tubule epithelial cells (HK-2 cell line) against TGHQ-induced toxicity. NAC provided remarkable protection against TGHQ-induced toxicity to HK-2 cells. NAC almost completely inhibited TGHQ-induced cell death, mitochondrial membrane potential collapse, as well as ROS production. NAC also attenuated TGHQ-induced DNA damage and the subsequent activation of poly (ADP-ribose) polymerase and ATP depletion. Moreover, NAC significantly attenuated c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase phosphorylation induced by TGHQ. In contrast, NAC itself markedly increased extracellular regulated kinase1/2 (ERK1/2) activation, and the upstream mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor, PD-98059, only partially inhibited this activation, suggesting that NAC can directly activate ERK1/2 activity. However, although NAC is frequently utilized as a glutathione (GSH) precursor, the cytoprotection afforded by NAC in HK-2 cells was not a consequence of increased GSH levels. We speculate that NAC exerts its protective effect in part by directly scavenging ROS and in part via ERK1/2 activation.

Chitosan oligosaccharides protect mice from LPS challenge by attenuation of inflammation and oxidative stress

Sepsis and its derivative syndromes are major causes of morbidity and mortality in the intensive care unit. Recently, lots of studies have shown that the progression of sepsis is attributed to redox imbalance and overproduction of proinflammatory cytokines. In previous studies, we have reported the anti-oxidative and anti-inflammatory effects of chitosan oligosaccharides in vitro. In the light of these findings, we applied the model of sepsis to mice by LPS injection to investigate whether chitosan oligosaccharides have a protective effect on LPS-induced sepsis. We found that treatment by chitosan oligosaccharides not only attenuated organ dysfunction but also improved survival rate after LPS injection. To further understand how it works, we examined several proinflammatory markers including neutrophil infiltration in organs and TNF-α and IL-1β in serum, and found that these cytokines were significantly reduced by chitosan oligosaccharide treatment. In addition to this, anti-oxidants including glutathione (GSH) and catalase (CAT) levels were depleted and malondialdehyde (MDA) levels were increased in LPS-induced sepsis, while chitosan oligosaccharides smoothed out the redox imbalance. Furthermore, we also assessed c-Jun NH 2-terminal kinase and p38 mitogen-activated protein kinase signal activation by LPS-stimulation, and found both of them were attenuated by chitosan oligosaccharide treatment. Collectively, our data demonstrated that chitosan oligosaccharides can protect mice from the LPS challenge by virtue of anti-inflammatory effects as well as anti-oxidation properties, which might offer beneficial effects for patients with sepsis.

Effects of Thymoquinone in the Expression of Mucin 4 in Pancreatic Cancer Cells: Implications for the Development of Novel Cancer Therapies

Pancreatic cancer is one of the most lethal cancers in the world, as it continues to be resistant to any therapeutic approaches. The high molecular weight glycoprotein mucin 4 (MUC4) is aberrantly expressed in pancreatic cancer and contributes to the regulation of differentiation, proliferation, metastasis, and the chemoresistance of pancreatic cancer cells. The absence of its expression in the normal pancreatic ductal cells makes MUC4 a promising target for novel cancer therapeutics. Natural products have been widely investigated as potential candidates in cancer therapies, and thymoquinone (TQ), extracted from the seeds of Nigella sativa, has shown excellent antineoplastic properties in some systems. In the present study, we evaluated the effect of TQ on pancreatic cancer cells and specifically investigated its effect on MUC4 expression. The MUC4-expressing pancreatic cancer cells FG/COLO357 and CD18/HPAF were incubated with TQ, and in vitro functional assays were done. The results obtained indicate that treatment with TQ downregulated MUC4 expression through the proteasomal pathway and induced apoptosis in pancreatic cancer cells by the activation of c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase pathways. In agreement with previous studies, the decrease in MUC4 expression correlated with an increase in apoptosis, decreased motility, and decreased migration of pancreatic cancer cells. MUC4 transient silencing studies showed that c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase pathways are activated in pancreatic cancer cells, indicating that the activation of these pathways by TQ is directly related to the MUC4 downregulation induced by the drug. Overall, TQ has potential for the development of novel therapies against pancreatic cancer.

RasGRP1 Is Essential for Ras Activation by the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate in Epidermal Keratinocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes and suggest JNK2 as one of the relevant downstream targets. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

The modulatory mechanisms of fenofibrate on human primary T cells

Fenofibrate is a lipid-lowering agent and supposed to have anti-inflammatory properties. But it was rarely evaluated for the signal transduction on human primary T cells. Therefore, the methods including enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay, luciferase assay and Western blotting were used to investigate the mechanisms of fenofibrate on human primary T cells, isolated from normal human beings. We found that fenofibrate could dose-dependently inhibit cytokine production such as interleukin-2, interleukin-4, tumor necrosis factor-alpha and interferon-gamma from activated T cells. Also, it could down-regulate activator protein-1 (AP-1) DNA-binding activities in T cells. As performing in vivo study, fenofibrate reduced the AP-1 transcriptional activity in Jurkat cells. Finally, fenofibrate inhibited the activation of c-Jun NH 2-terminal protein kinase and P38 mitogen-activated protein kinase. These results may extend potential therapeutic mechanisms of fenofibrate on cardiovascular disease with inflammatory processes.

Transcription Factor Egr-1 Is Essential for Maximal Matrix Metalloproteinase-9 Transcription by Tumor Necrosis Factor α

Matrix metalloproteinase-9 (MMP-9) is involved in a wide range of normal and pathologic conditions, including inflammation, tissue repair, tumor invasion, and metastasis. Tumor necrosis factor alpha (TNFalpha) is a major proinflammatory cytokine that plays crucial roles in tumor progression, including tumor invasion and metastasis in the tumor microenvironment. Egr-1 is a member of the zinc-finger transcription factor family induced by diverse stimuli, including TNFalpha. However, the role of Egr-1 in MMP-9 expression was previously unknown. This study shows that Egr-1 directly binds to the MMP-9 promoter and plays an essential role for TNFalpha induction of MMP-9 transcription. Furthermore, Egr-1 together with NF-kappaB can synergistically activate both basal and TNFalpha-induced MMP-9 promoter activities in the presence of p300. We found that Egr-1 mediates extracellular signal-regulated kinase and c-jun NH(2)-terminal kinase mitogen-activated protein kinase-dependent MMP-9 transcription on TNFalpha stimulation. The requirement for Egr-1 in MMP-9 expression is further supported by the fact that HeLa cells expressing Egr-1 siRNA and Egr-1-null mouse embryonic fibroblasts were refractory to TNFalpha-induced MMP-9 expression. This report establishes that Egr-1 is essential for MMP-9 transcription in response to TNFalpha within the tumor microenvironment.

Adipocyte Fatty Acid-binding Protein Modulates Inflammatory Responses in Macrophages through a Positive Feedback Loop Involving c-Jun NH2-terminal Kinases and Activator Protein-1

Adipocyte fatty acid-binding protein (A-FABP) has emerged as an important mediator of inflammation in macrophages. Macrophage-selective ablation of A-FABP alone is sufficient to prevent the development of high cholesterol diet-induced atherosclerosis in apoE-deficient mice. However, the precise mechanisms whereby A-FABP modulates inflammation remain elusive. Here, we report that A-FABP forms a finely tuned positive loop between JNK and activator protein-1 (AP-1) to exacerbate lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Real time PCR and luciferase reporter analysis showed that LPS induced A-FABP expression through transcriptional activation. This effect was mediated by JNK, which promoted the recruitment of c-Jun to a highly conserved AP-1 consensus binding motif located within the proximal region of the A-FABP promoter. LPS-induced transactivation of the A-FABP gene was diminished by either pharmacological inhibition of JNK or knocking down c-Jun or by mutating the AP-1 recognition site within the proximal region (-122 to -116 bp) of the A-FABP promoter. Conversely, the LPS-evoked phosphorylation of JNK, activation of AP-1, and production of pro-inflammatory cytokines were markedly attenuated by pharmacological or genetic suppression of A-FABP in macrophages. Furthermore, the LPS-induced elevation in A-FABP expression could also be prevented by the selective A-FABP inhibitor BMS309403. These findings support the notion that pharmacological inhibition of A-FABP represents a valid strategy for treating inflammation-related disorders such as atherosclerosis.

Proapoptotic Kinase MST2 Coordinates Signaling Crosstalk between RASSF1A, Raf-1, and Akt

Mammalian MST kinases function in stress-induced apoptosis to limit tumor progression. However, there is limited understanding about MST2 control by key regulators of cell division and survival. Raf-1 binds and inhibits MST2 kinase, whereas dissociation from Raf-1 and binding to tumor suppressor protein RASSF1A activates MST2. Akt phosphorylates MST2 in response to mitogens, oncogenic Ras, or depletion of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10. We identified T117 and T384 as Akt phosphorylation sites in MST2. Mutation of these sites inhibited MST2 binding to Raf-1 kinase but enhanced binding to tumor suppressor RASSF1A, accentuating downstream c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase signaling and promoting apoptosis. We determined that MST2 phosphorylation by Akt limits MST2 activity in two ways: first, by blocking its binding to RASSF1A and by promoting its association into the Raf-1 inhibitory complex, and second, by preventing homodimerization of MST2, which is needed for its activation. Dissociation of the Raf-1-MST2 complex promoted mitogenic signaling and coordinately licensed apoptotic risk. Using Ras effector domain mutants, we found that Akt is essential to prevent MST2 activation after mitogenic stimulation. Our findings elucidate how MST2 serves as a hub to integrate biological outputs of the Raf-1 and Akt pathways.

Mycophenolic acid attenuates tumor necrosis factor-α-induced endothelin-1 production in human aortic endothelial cells

AimsAtherosclerotic cardiovascular disease is the major cause of morbidity and mortality in solid organ transplant recipients. Endothelin-1 (ET-1) is implicated in the pathogenesis of atherosclerosis and is one of the potential therapeutic targets. This study was conducted to evaluate the effect of mycophenolic acid (MPA), an immunosuppressant for the transplant recipients, on tumor necrosis factor-α (TNF-α)-induced ET-1 production in aortic endothelial cells. Methods and resultsIn cultured human aortic endothelial cells, TNF-α increased ET-1 through AP-1 and NF-κB, whereas MPA attenuated it by reducing both AP-1 and NF-κB DNA-binding activities. TNF-α increased ET-1 via c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase. N-acetylcysteine that downregulated TNF-α-induced reactive oxygen species (ROS) inhibited JNK activation, but not p38 MAPK. N-acetylcysteine, SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated TNF-α-induced DNA-binding activities of both AP-1 and NF-κB. MPA inhibited JNK and p38 MAPK activations as well as ROS generation. N-acetylcysteine, SP600125, SB203580 and MPA had no effect on either TNF-α-induced IκBα degradation or p65 nuclear translocation, but attenuated p65 Ser276 phosphorylation. ConclusionMPA attenuated TNF-α-induced ET-1 production through inhibitions of ROS-dependent JNK and ROS-independent p38 MAPK that regulated NF-κB as well as AP-1. These findings suggest that MPA could have an effect of amelioration of atherosclerosis.

Basal c-Jun NH2-terminal protein kinase activity is essential for survival and proliferation of T-cell acute lymphoblastic leukemia cells

Hyperactivation of c-Jun NH2-terminal protein kinase (JNK) has been found in various malignant lymphocytes and inhibition of JNK activity leads to cell cycle arrest and apoptosis. However, the role of JNK activity in the oncogenic growth of T-cell acute lymphoblastic leukemia (T-ALL) cells remains largely unknown. Here, we report that treatment of T-ALL cells with JNK inhibitors led to cell cycle arrest and apoptosis and increased sensitivity to Fas-mediated apoptosis, whereas weak ectopic expression of MKK7-JNK1 fusion protein, which shows constitutive JNK activity, in T-ALL cells resulted in accelerated cell cycle progression and resistance to Fas-mediated apoptosis. The protein levels of c-Myc and Bcl-2 were reduced in the presence of JNK inhibitors but were enhanced with MKK7-JNK1. Small interfering RNA against JNK1, but not JNK2, exhibited similar effects to JNK inhibitors. These findings suggest that targeting JNK, especially JNK1 isoform, may have some important therapeutic implications in the treatment of T-ALL. Further exploration revealed that JNK protein and basal JNK activity in T-ALL cells showed aberrant subcellular localization, but no hyperactivation of JNK was observed. Thus, our work suggests that there might be novel mechanism(s) other than hyperactivation underlying the protumorigenic role of JNK activity.

Procaspase 8 and Bax Are Up-regulated by Distinct Pathways in Streptococcal Pyrogenic Exotoxin B-induced Apoptosis

We have previously identified integrin alpha(v)beta(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B that binds to Fas only. In the current study we found that after binding to alpha(v)beta(3), SPE B stimulated the tyrosine phosphorylation of JAK2 and STAT1. STAT1 tyrosine phosphorylation was inhibited by a JAK2 inhibitor, AG490, short interfering RNA (siRNA) silencing of JAK2, and anti-alpha(V)beta(3) antibody. AG490 also decreased the binding of tyrosine-phosphorylated STAT1 to the procaspase 8 promoter, decreasing procaspase 8 expression, suggesting that SPE B up-regulates procaspase 8 expression via the JAK2/STAT1 pathway. Alternatively, both SPE B and G308S increased STAT1 phosphorylation at serine 727, which was inhibited by anti-Fas antibody, a p38 inhibitor, SB203580, and siRNA silencing of p38. In addition, SPE B and G308S increased binding of serine-phosphorylated STAT1 to the Bax promoter and Bax expression, which was decreased by SB203580. SPE B and G308S-stimulated Bax expression was also inhibited by anti-Fas antibody. These findings suggest that Fas mediate SPE B-induced Bax expression through p38. Silencing of JAK2 or p38 by siRNA blocked procaspase 8 expression, whereas only p38 siRNA decreased Bax expression. Furthermore, JAK2 inhibition and p38 inhibition reduced SPE B-induced apoptosis, but only p38 inhibition blocked G308S-induced apoptosis.

Angiotensin II Regulates the Expression of Monocyte Chemoattractant Protein-1 in Pancreatic Cancer Cells

IntroductionPancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers with an overall median survival of less than 9 months and a 5-year survival rate of less than 5%. Increasing evidence indicates that inflammation facilitates PDA growth. DiscussionAngiotensin II (AngII), the principal hormone of the renin–angiotensin system, is actively generated in the pancreas and has been proposed as a key mediator of inflammation. Monocyte chemoattractant protein (MCP)-1 is a chemokine that plays an important role in the recruitment of mononuclear cells into sites of inflammation. In this study, we investigated the potential proinflammatory role of AngII in PDA through studying its effect on MCP-1. AngII significantly increased the expression of MCP-1 mRNA and protein in PDA cells and induced its promoter activity. Constitutive and AngII-induced MCP-1 transcription was inhibited by an AngII type 1 receptor (AT1R) blocker, but was unchanged by an AT2R blocker. AngII activated the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 or c-Jun NH2-terminal mitogen-activated protein kinases. Inhibition of ERK1/2 activation reduced the AngII-induced MCP-1 synthesis. AngII induced the activation and nuclear translocation of nuclear factor-κB (NF-κB), an effect that was inhibited by AT1R blockade. Inhibition of NF-κB by pyrrolidine dithiocarbamate decreased the AngII-mediated increase in MCP-1 mRNA. Our data provide a novel insight into an AngII-initiated signal transduction pathway that regulates MCP-1 as a possible inflammatory mechanism in PDA and suggest that AngII blockade may regulate chemokine-induced signal transduction to prevent or reduce inflammation in PDA.

Low concentration of arsenic could induce caspase-3 mediated head kidney macrophage apoptosis with JNK–p38 activation in Clarias batrachus

We had earlier demonstrated that chronic exposure (30 days) to micro-molar concentration (0.50 μM) of arsenic induced head kidney macrophage (HKM) death in Clarias batrachus. The purpose of the present study is to characterize the nature of HKM death induced by arsenic and elucidate the signal transduction pathways involved in the process. Arsenic-induced HKM death was apoptotic in nature as evident from DNA gel, Annexin V–propidium iodide, Hoechst 33342 staining and TdT-mediated dUTP nick end labeling (TUNEL) assays. Inhibitor studies and immunoblot analyses further demonstrated that arsenic-induced HKM apoptosis involved activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, a well-characterized caspase-3 substrate. Preincubation with antioxidants N-acetyl-cysteine or dimethyl sulfoxide significantly lowered reactive oxygen species (ROS) levels in arsenic-treated HKM and prevented caspase activation, malondialdehyde formation and HKM apoptosis. Arsenic induced membrane translocation of the NADPH oxidase subunit p47 phox . Preincubation with apocynin and diphenyleneiodonium chloride, both selective inhibitors of NADPH oxidases, prevented p47 phox translocation, ROS production and HKM death. Exposure of HKM to arsenic induced the activation of mitogen-activated protein kinase family (MAPK) proteins including c-Jun NH 2-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38). Preincubation of HKM with p38 inhibitor SB203580 and JNK inhibitor SP600125 protected the HKM against arsenic-induced apoptosis. We conclude that exposure to micro-molar concentration of arsenic induces ROS generation through the activation of NADPH oxidases, which in turn causes caspase-3 mediated HKM apoptosis. In addition, the study also indicates a role of p38–JNK pathway in arsenic-induced HKM apoptosis in C. batrachus.

Aldosterone-Induced Activation of Signaling Pathways Requires Activity of Angiotensin Type 1a Receptors

Aldosterone has been shown to induce vascular damage, endothelial dysfunction, and myocardial fibrosis, which depend in part on activation of angiotensin II (Ang II)-mediated pathways. However, mechanisms underlying crosstalk between Ang II type 1 receptor (AT(1)R) and mineralocorticoid receptor (MR) are mostly unknown. We tested whether the lack of Ang II type 1a receptor (AT(1a)R) or Ang II type 1b receptor (AT(1b)R) would decrease cellular effects induced by aldosterone. We examined the effect of Ang II or aldosterone after transfection of mesenteric vascular smooth muscle cells (VSMCs) from C57Bl/6 mice with small interference RNA for AT(1a)R, AT(1b)R, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-kappaB phosphorylation after a 20-minute stimulation. Small interference RNA for AT(1a)R downregulated phosphorylation of ERK1/2, JNK, and NF-kappaB after aldosterone stimulation compared to controls. Downregulation of AT(1b)R or MR only abolished the activation of NF-kappaB. In VSMCs from C57Bl/6 mice, aldosterone and Ang II induced the activation of the c-fos promoter from 30 minutes to 1 hour. This effect was blocked when using VSMCs from AT(1a)R knockout mice. We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT(1a)R and AT(1b)R. Our data suggest that aldosterone augments AT(1)R-dependent activation of ERK1/2, JNK, and NF-kappaB in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT(1)R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.

Activation Profile of Intracellular Mitogen-Activated Protein Kinases in Peripheral Lymphocytes of Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes. Abnormal activation of intracellular signaling molecules in lymphocytes by inflammatory cytokines can instigate the inflammation in SLE. The activation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in inflammatory cytokine IL-18-activated monocytes, CD4+ T helper (Th) lymphocytes, CD8+ T lymphocytes, and CD19+ B lymphocytes in 22 SLE patients and 20 sex- and age-matched control subjects were measured by flow cytometry. The basal expressions of phospho-p38 MAPK in CD4+ T lymphocytes, CD8+ T lymphocytes, and B lymphocytes were significantly higher in SLE patients than controls (all p<0.05). The expression of phospho-p38 MAPK in CD4+ T lymphocytes, CD8+ T lymphocytes and B lymphocytes, and phospho-JNK in CD8+ T lymphocytes and B lymphocytes was also significantly elevated in SLE patients upon the activation by IL-18, exhibiting significant correlation with the plasma concentrations of Th1 chemokine CXCL10 (all p<0.05). The expression of phospho-JNK in IL-18 activated CD8+ T lymphocytes and the relative % fold increase of the expression of phospho-JNK upon IL-18 activation in B lymphocytes were significantly correlated with SLE disease activity index (both p<0.05). The inflammation-mediated activation of JNK and p38 MAPK signaling pathways in T and B lymphocytes can be the underlying intracellular mechanisms causing lymphocyte hyperactivity in SLE.

Angiotensin II type-1 receptor regulates RhoA and Rho-kinase/ROCK activation via multiple mechanisms. Focus on “Angiotensin II induces RhoA activation through SHP2-dependent dephosphorylation of the RhoGAP p190A in vascular smooth muscle cells”

angiotensin ii, a crucial regulator of the cardiovascular system, is not only required for physiological functions, such as maintenance of blood pressure and plasma sodium concentration but also is involved in pathophysiological conditions such as hypertension and atherosclerosis (26). Angiotensin II exerts its effect by mainly binding to the angiotensin II type-1 (AT1) receptor. As its seven transmembrane structure suggests, the AT1 receptor couples to several heterotrimeric G proteins including Gq. In addition, the activation of the AT1 receptor leads to increased reactive oxygen species; activation of various protein tyrosine and serine/threonine kinases; and of small G proteins such as Ras, Rac, and RhoA, which may require heterotrimeric G protein-dependent and -independent mechanisms (6, 7, 11, 19).

PXII-13 Demonstration of chlamydia pneumoniae, mycoplasma pneumoniae, cytomegalovirus, and Epstein-Barr virus in atherosclerotic coronary arteries, in nonrheumatic calcific aortic valves and in rheumatic stenotic mitral valves by polymerase chain reaction

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl-p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH2-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77–1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.

Protective effect of histamine H 2 receptor antagonist ranitidine against rotenone-induced apoptosis

Histamine H 2 receptor antagonists have been reported to improve the motor symptoms of Parkinson's disease (PD) patients and to exert neuroprotective effects. In this study, we investigated the protective effects of the H 2 receptor antagonist ranitidine on rotenone-induced apoptosis in human dopaminergic SH-SY5Y cells, focusing on mitogen-activated protein kinases (MAPKs) and caspases (CASPs)-mediated apoptotic events. Ranitidine blocked the rotenone-induced phosphorylation of c-Jun NH 2-terminal protein kinase (JNK) and P38 MAPK (P38), and promoted the phosphorylation of extracellular signal-regulated protein kinase (ERK). Ranitidine also prevented the down-regulation of B-cell CLL/lymphoma 2 (BCL2) and the up-regulation of BCL2-associated X protein (BAX) by rotenone. Furthermore, ranitidine not only attenuated rotenone-induced cleavages of CASP9, poly(ADP-ribose) polymerase-1 (PARP) and CASP3, but also suppressed CASP3 enzyme activity. These results indicate that ranitidine protects against rotenone-induced apoptosis, inhibiting phosphorylation of JNK and P38, and activation of CASPs in human dopaminergic SH-SY5Y cells.

Role for RACK1 Orthologue Cpc2 in the Modulation of Stress Response in Fission Yeast

The receptor of activated C kinase (RACK1) is a protein highly conserved among eukaryotes. In mammalian cells, RACK1 functions as an adaptor to favor protein kinase C (PKC)-mediated phosphorylation and subsequent activation of c-Jun NH(2)-terminal kinase mitogen-activated protein kinase. Cpc2, the RACK1 orthologue in the fission yeast Schizosaccharomyces pombe, is involved in the control of G2/M transition and interacts with Pck2, a PKC-type protein member of the cell integrity Pmk1 mitogen-activated protein kinase (MAPK) pathway. Both RACK1 and Cpc2 are structural components of the 40S ribosomal subunit, and recent data suggest that they might be involved in the control of translation. In this work, we present data supporting that Cpc2 negatively regulates the cell integrity transduction pathway by favoring translation of the tyrosine-phosphatases Pyp1 and Pyp2 that deactivate Pmk1. In addition, Cpc2 positively regulates the synthesis of the stress-responsive transcription factor Atf1 and the cytoplasmic catalase, a detoxificant enzyme induced by treatment with hydrogen peroxide. These results provide for the first time strong evidence that the RACK1-type Cpc2 protein controls from the ribosome the extent of the activation of MAPK cascades, the cellular defense against oxidative stress, and the progression of the cell cycle by regulating positively the translation of specific gene products involved in key biological processes.

Regulation of Bax by c-Jun NH 2-terminal kinase and Bcl-xL in vinblastine-induced apoptosis

Microtubule inhibitors, such as vinblastine, are widely used in cancer chemotherapy. Vinblastine exerts its antitumor effect by inducing apoptosis. In KB-3 cells, we have shown previously that vinblastine activates c-Jun NH 2-terminal protein kinase (JNK) and causes Bax mitochondrial translocation and activation. In this study, we sought to test the hypothesis that JNK and Bcl-xL act as positive and negative regulators, respectively, of Bax translocation. The JNK inhibitor SP600125 inhibited vinblastine-induced JNK activation and in concert inhibited Bax mitochondrial translocation, Bax oligomerization, and Bax activation. Furthermore, the JNK inhibitor blocked vinblastine-induced apoptosis. The ability of vinblastine to induce Bax translocation and the inhibitory effect of SP600125 were confirmed in cells stably expressing GFP-Bax. However, if transiently overexpressed, Bax localized to the mitochondria, and this was associated with loss of viability and subsequent cell death. If Bcl-xL was co-expressed with Bax, the cells readily tolerated Bax overexpression. Indeed, physical interaction between Bcl-xL and Bax but not Bak was demonstrated by co-immunoprecipitation. These findings provide novel insight into the role of Bax and its regulation in vinblastine-induced apoptosis.