Kavalactones protect neural cells against amyloid beta peptide-induced neurotoxicity via extracellular signal-regulated kinase 1/2-dependent nuclear factor erythroid 2-related factor 2 activation

One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide (AP), which can initiate a cascade of oxidative events that may result in neuronal death. Because nuclear factor erythroid 2-related factor 2 (Nrf2) is the major regulator for a battery of genes encoding detoxifying and antioxidative enzymes via binding to the antioxidant response element (ARE), it is of great interest to find nontoxic activators of Nrf2 rendering neuronal cells more resistant to AP toxicity. Using ARE-luciferase assay and Western blot, we provide evidence that the kavalactones methysticin, kavain, and yangonin activate Nrf2 time- and dose-dependently in neural PC-12 and astroglial C6 cells and thereby up-regulate cytoprotective genes. Viability and cytotoxicity assays demonstrate that Nrf2 activation is able to protect neural cells from amyloid beta-(1-42) induced neurotoxicity. Down-regulation of Nrf2 by small hairpin RNA as well as extracellular signal-regulated kinase 1/2 inhibition abolishes cytoprotection. We further give evidence that kavalactone-mediated Nrf2 activation is not dependent on oxidative stress production. Our results demonstrate that kavalactones attenuate amyloid beta-peptide toxicity by inducing protective gene expression mediated by Nrf2 activation in vitro. These findings indicate that the use of purified kavalactones might be considered as an adjunct therapeutic strategy to combat neural demise in Alzheimer disease and other oxidative stress-related diseases.     

Promiscuous coupling and involvement of protein kinase C and extracellular signal-regulated kinase 1/2 in the adenosine A1 receptor signalling in mammalian spermatozoa

Mammalian spermatozoa require a maturational event after ejaculation that allows them to acquire the capacity for fertilisation. This process occurs spontaneously during the transit through the female reproductive tract where spermatozoa are in contact with micromolar concentrations of adenosine that might act as a capacitative effector. This study shows that the adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine, can induce capacitation, i.e., the ability to undergo the acrosome reaction and to become fertile. This receptor, already known to be bound to Galpha(i2), is also bound to G(q/11). These G proteins are functional in the signalling pathway elicited by the A1 receptor and correlate with the multiple intracellular events that follow its activation. The use of protein kinase C isoform inhibitors and MEK inhibitors, resulting in the abolition of the biological response to the selective agonist, indicates the involvement of protein kinase C and MEK in its signalling. In agonist-treated spermatozoa an extracellular calcium influx, involvement of alpha and gamma PKC isoforms and transient phosphorylation of ERK1/2 have been observed. Our results, besides showing that adenosine A1 receptor prompts mammalian spermatozoa to undergo the acrosome reaction hence supporting a role for adenosine as agent for fertilisation, show that 2-chloro-N6-cyclopentyladenosine triggers signalling mechanisms that involve both Galpha(i2) and G(q/11), extracellular calcium influx, modulation of classical Ca2+-dependent PCK isoforms and up-regulation of the ERK1/2 phosphorylation.     

The noble gas argon modifies extracellular signal-regulated kinase 1/2 signaling in neurons and glial cells

Recently, the noble gas argon has been identified as a potent neuroprotective agent, but little is known about its cellular effects. In this in vitro study, we investigated argon's influence on the extracellular signal-regulated kinase (ERK) 1/2, a ubiquitous enzyme with numerous functions in cell proliferation and survival. Primary neuronal and astroglial cell cultures and the microglial cell line BV-2 were exposed to 50 vol.% argon. Further possible effects were studied following stimulation of microglia with 50 ng/ml LPS. ERK 1/2 activation was assessed by phosphorylation state-specific western blotting, cytokine levels by real-time PCR and western blotting. Total phosphotyrosine phosphatase activity was examined with p-nitrophenylphosphate. After 30 min exposure, argon significantly activated ERK 1/2 signaling in microglia. Enhanced phosphorylation of ERK 1/2 was also found in astrocytes and neurons following argon exposure, but it lacked statistical significance. In microglia, argon did not substantially interfere with LPS-induced ERK1/2 activation and inflammatory cytokine induction. Addition of the MEK-Inhibitor U0126 abolished the induced ERK 1/2 phosphorylation. Cellular phosphatase activity and the inactivation of phosphorylated ERK 1/2 were not altered by argon. In conclusion, argon enhanced ERK 1/2 activity in microglia via the upstream kinase MEK, probably through a direct mode of activation. ERK 1/2 signaling in astrocytes and neurons in vitro was also influenced, although not with statistical significance. Whether ERK 1/2 activation by argon affects cellular functions like differentiation and survival in the brain in vivo will have to be determined in future experiments.     

Dissecting the roles of checkpoint kinase 1/CDC2 and mitogen-activated protein kinase kinase 1/2/extracellular signal-regulated kinase 1/2 in relation to 7-hydroxystaurosporine-induced apoptosis in human multiple myeloma cells

The functional roles of Cdc2 and checkpoint kinase 1 (Chk1) in synergistic interactions between 7-hydroxystaurosporine (UCN-01) and mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors [e.g., 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide (PD184352)] were examined in human multiple myeloma cells in relation to MEK1/2/ERK1/2 activation and lethality. Time course studies revealed that MEK1/2/extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation preceded Cdc2 dephosphorylation (Tyr15) after UCN-01 exposure. Furthermore, enforced expression of Cdc2 or small inducible RNA (siRNA)-mediated Cdc2 knockdown failed to modify ERK1/2 activation status in either the presence or absence of UCN-01, arguing against a causal relationship between these events. However, ectopic expression of Cdc2 sensitized cells to the lethality of UCN-01/MEK inhibitor regimen, whereas Cdc2 knockdown by siRNA significantly diminished the lethal effects of this combination. Conversely, Chk1 knockdown by siRNA enhanced lethality mediated by UCN-01/PD184352. It is interesting that Chk1 knockdown reduced basal ERK1/2 activation and antagonized the ability of UCN-01 to activate ERK1/2. Finally, ectopic expression of constitutively active MEK1 significantly protected cells from the UCN-01/MEK1/2 inhibitor regimen without modifying Cdc2 activation status. Together, these findings indicate that although UCN-01-mediated Chk1 inhibition and Cdc2 activation are unlikely to be responsible for MEK1/2/ERK1/2 activation, both of these events contribute functionally to enhanced lethality in cells coexposed to MEK inhibitors. They also suggest a role for Chk1 in UCN-01-induced ERK1/2 activation, implying the existence of a heretofore unrecognized link between Chk1 and ERK1/2 signaling.     

Protective effects of betulin and betulinic acid against ethanol-induced cytotoxicity in HepG2 cells

Plant triterpenes, such as oleanolic acid and betulin were described as hepatoprotectants active against cytotoxicity of acetaminophen or cadmium. The aim of this paper is to compare the cytoprotective activity of betulin, betulinic acid and oleanolic acid against ethanol-induced cytotoxicity in HepG2 cells. The influence of three triterpenes on ethanol-induced production of superoxide anion and hydrogen peroxide was also examined. Among the examined triterpenes, betulin was the most active protectant of HepG2 cells against ethanol-induced cytotoxicity. Betulin and betulinic acid significantly decreased ethanol-induced production of superoxide anion. Oleanolic acid inhibited only ethanol- and phorbol ester-induced production of hydrogen peroxide. The results indicate that cytoprotective or antioxidative activity of triterpenes depends on their chemical structure.     

Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cells via extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.     

Protective effects of triterpene compounds against the cytotoxicity of cadmium in HepG2 cells

The effects of triterpene compounds on cadmium toxicity were investigated in HepG2 cells. Ten triterpene compounds were examined, namely, betulin, soyasapogenol A, soyasapogenol B, ursolic acid, uvaol, oleanolic acid, friedelin, glycyrrhizin, 18alpha-glycyrrhetinic acid, and 18beta-glycyrrhetinic acid, and betulin, soyasapogenol A, and uvaol were found to reduce the toxicity of CdCl(2). In particular, betulin almost completely abolished the cytotoxicity of CdCl(2) at concentrations as low as 0. 1 microg/ml. The effects of betulin were particularly apparent when added to the culture medium before the addition of CdCl(2). Moreover, when HepG2 cells were incubated with betulin and then incubated in fresh betulin-free medium before the addition of CdCl(2), the toxic effects of cadmium were reduced. Betulin had no significant effect on the intracellular accumulation of cadmium, nor did it bind to cadmium, at least not in a test tube. When HepG2 cells were treated first with cycloheximide or actinomycin D, the subsequent protective effect of betulin against cadmium toxicity was significantly reduced, suggesting that betulin might protect cells against cadmium toxicity by inducing the synthesis of a certain protein or proteins. The synthesis of metallothionein, a protein that is known to reduce the toxicity of heavy metals, was not induced by betulin. However, using the differential display method, we confirmed that betulin promoted the expression of several genes. Our findings suggest that betulin might reduce cadmium toxicity by promoting the synthesis of certain proteins that protect cells against the toxic effects of cadmium.     

Tanshinone I enhances learning and memory,and ameliorates memory impairment in mice via the extracellular signal-regulated kinase signalling pathway

Background and purpose:

The intracellular signalling kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) is required for new memory formation, suggesting that control of ERK signalling might be a target for the treatment of cognitive dysfunction. Previously, we reported that tanshinone congeners have ameliorating effects on drug-induced memory impairment in mice. Here, we have investigated possible modes of action of tanshinone I on learning and memory, associated with ERK phosphorylation.

Experimental approach:

Using immunohistochemical, Western blot techniques, and behavioural testing, we studied the effect of tanshinone I on memory impairment induced by diazepam or dizocilpine (MK-801) in mice.

Key results:

Tanshinone I (2 or 4 mg·kg⁻¹, p.o.) increased latency times versus vehicle-treated control group in the passive avoidance task. Western blot analysis and immunohistochemical data showed that tanshinone I (4 mg·kg⁻¹) increased levels of phosphorylated cAMP response element binding protein (pCREB) and phosphorylated ERK (pERK) in the hippocampus. These increases in pCREB and pERK were blocked by U0126 (inhibitor of ERK1/2), which also prevented the increase in passive avoidance task latency time after tanshinone I. In models of learning and memory impairment induced by diazepam and MK-801, tanshinone I (4 mg·kg⁻¹) reversed learning and memory impairments detected by the passive avoidance test. Western blot analysis showed that tanshinone I reversed the diazepam- and MK-801-induced inhibitions of ERK and CREB activation in hippocampal tissues. These effects were also blocked by U0126.

Conclusions and implications:

Tanshinone I ameliorates the learning and memory impairments induced by diazepam and MK-801 through activation of ERK signalling.     

Elastin peptides activate extracellular signal-regulated kinase 1/2 via a Ras-independent mechanism requiring both p110gamma/Raf-1 and protein kinase A/B-Raf signaling in human skin fibroblasts

Elastin peptides (EPs) produced during cancer progression bind to the elastin binding protein (EBP) found at the surface of dermal fibroblasts, leading to the expression of collagenase-1 gene. The production of this enzyme involved in stromal reaction is caused by the sustained activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway via cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K). However, the mechanism of these signaling events remains unknown. We show that kappa-elastin (kappaE), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) and 2-(4-morpholynil)-8-phenyl-4H-1-bemzopyran-4-one (LY294002), respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. kappaE induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110gamma is expressed and activated by betagamma-derived subunits from a pertussis toxin-sensitive G protein after fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. In addition, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner after kappaE treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by kappaE was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Ras-independent ERK1/2 activation system in which p110gamma/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110gamma and B-Raf seem to be important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.     

dl-3n-Butylphthalide promotes angiogenesis via the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase signaling pathways

We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.     

Porphyromonas gingivalis, Periodontal pathogen, lipopolysaccharide induces angiogenesisvia extracellular signal-regulated kinase 1/2 activation in human vascular endothelial cells

Porphyromonas gingivalis is a major periodontal pathogen. The lipopolysaccharide (LPS) secreted from P. gingivalis is implicated in the initiation and progression of periodontitis. Aberrant angiogenesis is often associated with lesion formation in chronic periodontitis. In this study, we report that P. gingivalis LPS activates angiogenic cascade, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs). Furthermore, P. gingivalis LPS potently stimulated in vivo neovascularization in chick chorioallantoic membrane (CAM) and the mouse Matrigel plug assay. P. gingivalis LPS had no effect on the expression of vascular endothelial growth factor (VEGF) or its receptor, Flk-1, implying that P. gingivalis LPS-induced angiogenesis may result from its direct action on endothelial cells. P. gingivalis LPS evoked activation of the mitogen-activated protein kinase ERK1/2 in HUVECs, which is closely linked to angiogenesis. Taken together, these results strongly suggest P. gingivalis LPS plays an important role in the pathological angiogenesis for periodontal diseases, such as periodontitis.     

Inhibition of extracellular signal-regulated kinase (ERK1/2) activity reverses endotoxin-induced hypotension via decreased nitric oxide production in rats.

Overproduction of reactive oxygen and nitrogen species leads to oxidative stress and decreased total antioxidant capacity, which is responsible for high mortality from several inflammatory diseases such as endotoxic shock. Among reactive nitrogen species, nitric oxide (NO) produced by inducible NO synthase (iNOS) during endotoxemia is the major cause of vascular hyporeactivity, hypotension and multiple organ failure. This study was conducted to determine if mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK1/2) contributes to endotoxin-induced hypotension as well as vascular inflammation and oxidative stress via NO production. In conscious male Wistar rats, injection of endotoxin (10 mg kg(-1), i.p.) caused a decrease in mean arterial pressure (MAP) for 4h and increased levels of nitrite in serum, aorta and mesenteric artery. These effects of endotoxin were prevented by selective inhibition of ERK1/2 phosphorylation by MAPK kinase (MEK1/2) with U0126 (5 mg kg(-1), i.p.; 1h after endotoxin). Endotoxin also caused an increase in protein levels of phosphorylated ERK1/2 in aorta which was abolished by U0126. Selective inhibition of iNOS with phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide (1,3-PBIT) (10 mg kg(-1), i.p.; 1h after endotoxin) did not change the endotoxin-induced increase in ERK1/2 activity. Myeloperoxidase activity was increased in aorta and decreased in mesenteric artery by endotoxin, which was reversed by U0126. Endotoxin-induced decrease in one of the products of lipid peroxidation, malonedialdehyde (MDA) was prevented by U0126 in mesenteric artery; however, U0126 caused a further decrease in the levels of MDA in aorta. These data suggest that increased phosphorylation of ERK1/2 by MEK1/2 contributes to the endotoxin-induced hypotension via NO production rat aorta and mesenteric artery. It is likely that ERK1/2 mediates the effect of endotoxin on MPO activity in a different degree in the tissues suggesting possible involvement of any mediator and/or mechanism which also causes neutrophil infiltration during inflammatory response at least in mesenteric artery. Moreover, ERK1/2 seems to be involved in the endotoxin-induced increase in total antioxidant capacity in mesenteric artery.     

Combined effect of protein kinase B inhibitor or extracellular signal-regulated kinase inhibitor against farnesyltransferase inhibition-induced apoptosis in SiHa cells

The present study investigated the combined effect of Akt or extracellular signal-regulated kinase (ERK) inhibition in the presence of farnesyltransferase inhibitor against human cervix and uterus tumor cell line SiHa cells. Farnesyltransferase inhibitor may induce apoptosis through the mitochondria-mediated process and inhibition of the MEK, ERK, and Akt activity. Inhibitors of Akt and ERK at low concentrations seem to prevent the farnesyltransferase inhibitor-induced apoptosis in cervical SiHa cells by suppressing the mitochondrial membrane permeability change that leads to cytochrome c release and caspase-3 activation. These effects may be associated with inhibition of the reactive oxygen species formation and glutathione depletion. In contrast, at higher concentrations more than 1 muM, the Akt inhibitor and ERK inhibitor seem to exhibit an additive toxic effect against farnesyltransferase inhibitor-induced apoptosis by increasing mitochondrial membrane permeability change and oxidative stress, which may not involve inhibition of MEK, ERK, and Akt activity.     

Protective effects of protopine on hydrogen peroxide-induced oxidative injury of PC12 cells via Ca(2+) antagonism and antioxidant mechanisms

Calcium and lipid peroxidation play important roles in oxidative stress-induced cellular injury and apoptosis, which ultimately cause cell death. In this study we examined whether protopine had a neuroprotection against H₂O₂-induced injury in PC12 cells. Pretreatment of PC12 cells with protopine improved the cell viability, enhanced activities of superoxide dismutase, glutathione peroxidase and catalase, and decreased malondialdehyde level in the H₂O₂ injured cells. Protopine also reversed the increased intracellular Ca²⁺ concentration and the reduced mitochondrial membrane potential caused by H₂O₂ in the cells. Furthermore, protopine was able to inhibit caspase-3 expression and cell apoptosis induced by H₂O₂. In summary, this study demonstrates that protopine is able to relieve H₂O₂-induced oxidative stress and apoptosis in PC12 cells, at least in part, by Ca²⁺ antagonism and antioxidant mechanisms.