Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways: role of c-Jun N-terminal kinase in paraquat-induced cell death.

Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP(+)), a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB) activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 had no effect on paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented clearly suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and support the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment.     

Andrographolide acts through inhibition of ERK1/2 and Akt phosphorylation to suppress chemotactic migration

We now evaluated the anti-inflammatory mechanisms of andrographolide on complement 5a (C5a)-induced macrophage recruitment in vitro. Andrographolide concentration dependently inhibited cell migration toward C5a with an IC50 of 5.6+/-0.7 microM. With relatively specific kinase inhibitors (PD98059, SB203580, SP600125, wortmannin and LY294002, respectively) the results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol-3-kinase (PI3K) were necessary for C5a-induced migration, whereas c-Jun N-terminal kinase (JNK) was nonessential. Andrographolide significantly attenuated C5a-stimulated phosphorylation of ERK1/2, and of its upstream activator, MAP kinase-ERK kinase (MEK1/2). C5a-activated ERK1/2 phosphorylation was 86+/-9% inhibited by 30 microM andrographolide. Under the same conditions, however, andrographolide failed to affect C5a-stimulated p38 MAPK and JNK phosphorylation. Andrographolide also strongly abolished C5a-stimulated Akt phosphorylation, a downstream target protein for PI3K. These results indicate that inhibition of cell migration by interfering with ERK1/2 and PI3K/Akt signal pathways may contribute to the anti-inflammatory activity of andrographolide.     

Ebselen inhibits p38 mitogen-activated protein kinase-mediated endothelial cell death by hydrogen peroxide

Ebselen (2-phenyl-1, 2-benzisoselenazol-3[2H]-one) is a seleno-organic compound exhibiting both glutathione peroxidase and antioxidant activity. Although it has been reported that ebselen is effective against hydrogen peroxide (H(2)O(2))-induced cell death in several cell types, its effect on endothelial cell damage has not yet been elucidated. In the present study, we examined the effect of ebselen on H(2)O(2)-induced human umbilical vein endothelial cells (HUVECs) death, and its intracellular mechanism. Our findings showed that pretreatment of HUVECs with ebselen resulted in a significant recovery from H(2)O(2)-induced cell death in a concentration-dependent manner. In addition to the inhibition of lactate dehydrogenase (LDH) leakage, ebselen inhibited H(2)O(2)-induced cytochrome c release and caspase-3 activation and the resultant apoptosis in HUVECs. Moreover, it was observed that H(2)O(2) significantly stimulated activation of mitogen-activated protein (MAP) kinases, i.e., p38 MAP kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2). Ebselen inhibited H(2)O(2)-induced p38 MAP kinase, but not JNK or ERK1/2 activation. Furthermore, SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]-1H-imidazole), a specific p38 MAP kinase inhibitor, inhibited H(2)O(2)-induced p38 MAP kinase phosphorylation, cytochrome c release, caspase-3 activation, as well as cell death in HUVECs. These findings suggest that ebselen attenuates H(2)O(2)-induced endothelial cell death through the inhibition of signaling pathways mediated by p38 MAP kinase, caspase-3, and cytochrome c release. Thus, inhibition of p38 MAP kinase by ebselen may imply its usefulness for prevention and/or treatment of endothelial cell dysfunction, which was suggested to be the first step in the development of atherosclerosis.     

Potentiation of arsenic-induced cytotoxicity by sulfur amino acid deprivation (SAAD) through activation of ERK1/2, p38 kinase and JNK1: the distinct role of JNK1 in SAAD-potentiated mercury toxicity

Sulfur amino acid deficiency occurs in certain pathophysiological situations (e.g. protein-calorie malnutrition). Previous studies revealed that sulfur amino acid deprivation (SAAD) activated MAP kinases and potentiated cadmium-induced cytotoxicity by activation of ERK1/2 in conjunction with p38 kinase or JNK. The present study was designed to determine susceptibility of cells to a variety of heavy metals in combination with SAAD. Viability was assessed in H4IIE cells treated with sodium arsenite, mercuric chloride, sodium selenite, lead acetate, chromium trioxide or manganese chloride. SAAD potentiated the cytotoxicity of H4IIE cells by arsenic or mercury (i.e. EC50, 19 and 5 microM in SAAD vs. 401 and 42 microM in control medium, respectively). TUNEL assays revealed that the potentiated arsenic or mercury toxicity involved apoptotic cell death. Lead or selenite moderately elicited cell death, which was not enhanced by SAAD. Chromium or manganese caused no significant cytotoxicity. Treatment of cells with U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] an ERK1/2 inhibitor or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] a p38 kinase inhibitor effectively prevented SAAD-potentiated arsenic toxicity. The potentiated arsenic toxicity was also inhibited in cells stably expressing a dominant negative mutant of c-Jun N-terminal kinase 1 [JNK1(-)]. The inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase failed to prevent mercury-induced toxicity enhanced by SAAD. JNK1(-) cells were minimally susceptible to mercury in SAAD medium. These results demonstrated that SAAD potentiated cytotoxicity induced by arsenic or mercury and that activation of ERK1/2, p38 kinase and JNK1 was responsible for the potentiated arsenic toxicity, whereas the mercury toxicity enhanced by SAAD was mediated with the activity of JNK1.     

Monocyte-mediated rotenone neurotoxicity towards human neuroblastoma SH-SY5Y: role of mitogen-activated protein kinases

Increasing evidence has suggested an important role for rotenone in the pathogenesis of Parkinson's disease (PD). In this report, sequential linking of two culture systems, monocytic THP-1 cell line and SH-SY5Y neuroblastoma, was utilized. The supernatant from rotenone-stimulated THP-1 cells was used as the incubating medium for the second culture which adopted cells of the SH-SY5Y neuroblastoma. At 6.25-50 nM, concentrations that were nontoxic to SH-SY5Y directly, rotenone induced dose-dependent cell death on SH-SY5Y through stimulating monocyte THP-1 within a period of 48 h. Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Hoechst 33258 staining revealed that the treatment of SH-SY5Y with rotenone-stimulated THP-1 supernatant resulted in condensed nuclei and a decrease in cell size. Apoptotic rate measured by flow cytometric analysis indicated that at 25 and 50 nM, the percentage of apoptotic SH-SY5Y cells accumulated to 31.5% and 37.0% respectively. We further investigated whether rotenone (50 nM) activated mitogen-activated protein kinase (MAPK) cascades, and found it had effect on p38 MAPK and ERK in THP-1 cells, but not JNK. Pretreatment of THP-1 cells with the MAPK kinase inhibitor, PD98059, inhibited THP-1 cell-mediated rotenone neurotoxicity towards SH-SY5Y, whereas the p38 MEK inhibitor, SB203580, had no effect. These results suggested that activation of microglia intracellular signaling pathway may also involve in microglia-enhanced rotenone neurotoxicity.     

Differentiation-inducing factor-1-induced growth arrest of K562 leukemia cells involves the reduction of ERK1/2 activity

The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell differentiation in the cellular slime mold Dictyostelium discoideum. In addition, DIF-1 is a potent antileukemic agent that induces growth arrest in K562 cells. In this study, we investigated the mechanism of action of DIF-1 in K562 cells in the light of cell-cycle regulators such as cyclins, retinoblastoma protein (pRb), and the mitogen-activated protein kinase (MAPK) family. DIF-1 down-regulated cyclins D/E and a phosphorylated form of pRb (p-pRb), and thereby induced G(1) arrest of the cell cycle. DIF-1 inactivated the extracellular signal-regulated kinase (ERK) in a biphasic manner but did not affect the c-Jun N-terminal kinase (JNK) or p38 MAPK. The MEK (MAPK kinase) inhibitor, U0126, which has been shown to induce growth arrest, inactivated ERK and down-regulated cyclins D and E. Although DIF-1 activated the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway, neither wortmannin nor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002; PI-3K inhibitors) cancelled DIF-1-induced growth arrest. The present results suggest that ERK inactivation may be involved in DIF-1-induced growth arrest and that PI-3K activity is not required for DIF-1-induced growth arrest in K562 cells.     

p38 and JNK MAPK, but not ERK1/2 MAPK, play important role in colchicine-induced cortical neurons apoptosis

Colchicine is a microtubule interfering agent and is able to induce neural apoptosis. However, the intracellular pathway involved in its neurotoxicity is still unclear. In the present study, three of mitogen-activated protein kinases (MAPKs): p38, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase 1/2 (ERK1/2) were investigated in colchicine-induced apoptosis on cortical neurons for the first time. Our results showed that 1 microM colchicine administration in primarily cultured cortical neurons led to typical neuronal apoptosis, and the apoptosis was attenuated by taxol, a microtubule stabilizer. Moreover, activation of p38 MAPK was found for the first time, as well as that of JNK MAPK, but not of ERK1/2 MAPK, after colchicine exposure. Apoptosis was inhibited by p38 MAPK inhibitors, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole), SB239063 (trans-1-(4-hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxypyrimidin-4-yl) imidazole), and JNK MAPK pathway inhibitors, CEP11004 (9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-5,16-bis[[(1-methylethyl)thio]methyl]-1-oxo-, methyl ester, (9S,10R,12R)-), SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). However, PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) and U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene), ERK1/2 MAPK inhibitors, did not work. Furthermore, better neuronal protective effects were achieved by using JNK and the p38 MAPK inhibitors together as compared to that by using either alone. The results suggested that p38 MAPK, JNK MAPK, but not ERK1/2 MAPK may play pivotal role in colchicine's neurotoxicity in primarily cultured cortical neurons, and the protective effects of the inhibition of p38 or JNK MPAK on cortical neurons were synergistically.     

Involvement of the extracellular signal-regulated protein kinase (ERK) pathway in the induction of apoptosis by cadmium chloride in CCRF-CEM cells

When CCRF-CEM cells were incubated with 5–40 μM CdCl_2, apoptosis was observed most clearly at 10 μM. Prior to the development of apoptosis, mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, were activated with different sensitivity to CdCl₂ exposure. ERK and p38 MAPK were phosphorylated with incubation of 1 μM CdCl_2, but higher than 20 μM CdCl₂ was required for the clear phosphorylation of JNK. In the time–course study, ERK and p38 MAPK were phosphorylated earlier than JNK after CdCl₂ exposure. The in vitro activities of MAPKs also increased in response to CdCl₂ exposure. Pretreatment with an intracellular Ca²⁺ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), suppressed almost completely CdCl₂-induced phosphorylation of JNK and p38 MAPK, but not ERK phosphorylation, indicating that the activation of JNK and p38 MAPK depends on the intracellular Ca²⁺ but that of ERK does not. On the other hand, treatment with a MAPK/ERK kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), suppressed CdCl₂-induced ERK activation and the apoptosis as well. The inhibition of p38 MAPK activity with SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H-imidazole) did not protect cells from apoptosis. The present results showed that the activation of ERK, JNK, and p38 MAPK is differently regulated in CCRF-CEM cells exposed to CdCl_2, and that the ERK pathway seems to be responsible for the induction of apoptosis by CdCl₂ exposure in this human T cell line.     

C5a differentially stimulates the ERK1/2 and p38 MAPK phosphorylation through independent signaling pathways to induced chemotactic migration in RAW264.7 macrophages

We elucidate the roles of various protein kinases involved in complement 5a (C5a)-induced cell migration. Results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (P13K) were necessary for C5a-induced migration, whereas protein kinase C and c-Jun N-terminal kinase (JNK) were nonessential. C5a-induced migration was also suppresses by phospholipase C (PLC) inhibitor U73122 and pertussis toxin (PTX). We found that C5a-induced, time-dependent (1) ERK1/2 phosphorylation was markedly diminished by PTX, U73122, P13K inhibitors wortmannin and LY294002 and ERK1/2 inhibitor PD98059; (2) Akt phosphorylation was also attenuated by the above inhibitors except PD98059; (3) p38 MAPK phosphorylation was only affected by PTX. Furthermore, C5a also stimulated PLCbeta(2) membrane translocation in a time-dependent manner that occurred early prior to Akt phosphorylation and could be abolished only by PTX and U73122. These results suggest that C5a, through the activation of PTX-sensitive G protein, to differentially stimulate ERK1/2 and p38 MAPK phosphorylation and evoke cell migration. That is, ERK1/2 but not p38 MAPK phosphorylation is down stream of P13K/Akt and modulated by PLC. Additionally, beta(2) isoform may be one of the participates in C5a signal and acts more upstream of P13K/Akt.     

Eupatilin inhibits H2O2-induced apoptotic cell death through inhibition of mitogen-activated protein kinases and nuclear factor-κB

Eupatilin (5,7-dihydroxy-3',4',6-trimethoxyflavone), an extract from Artemisia asiatica Nakai, is a flavonoid of pharmacologically active ingredients. Eupatilin is known to possess anti-cancer, anti-inflammatory, and anti-oxidative activity. Recently, eupatilin has been reported to be effective in producing gastric mucosal as an anti-gastritis agents. However, the mechanism of protective action is still unknown. We studied cytoprotective actions of eupatilin on H(2)O(2)-induced cell death and its possible mechanisms of action in human gastric (AGS) cells. Eupatilin dose-dependently inhibited H(2)O(2)-induced apoptosis as indicated by co-staining with Annexin V and propidium iodide. Hydrogen peroxide provoked phosphorylation of extracellular regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK), and activation of nuclear factor-kappaB (NF-kappaB). On the contrary, eupatilin decreased H(2)O(2)-induced activation of ERK, JNK and NF-kappaB. In addition, treatment of specific inhibitors for ERK, JNK, and NF-kappaB attenuated H(2)O(2)-induced apoptosis. Co-treatment of inhibitors and eupatilin was more effective in decreasing H(2)O(2)-induced apoptosis. Taken together, we suggest that eupatilin inhibits H(2)O(2)-induced apoptosis through the inhibition ERK, JNK, and NF-kappaB.     

Alterations of carbohydrate and lipid intermediary metabolism during inhibition of glucose-6-phosphatase in rats

Adrenomedullin is a potent vasodilatory peptide that has a variety of effects in a number of different systems including kidney. In cultured rat glomerular mesangial cells adrenomedullin increases cAMP, decreases proliferation and increases apoptosis. Associated with the anti-proliferative and apoptotic effects, adrenomedullin also causes a decrease in extracellular signal-regulated kinase2 (ERK2) and an increase in cJun N-terminal kinase 1 (JNK1) and P38 mitogen-activated protein kinase (P38 MAPK) activities. The purpose of the present study was to examine the role of P38 MAPK on adrenomedullin-mediated inhibition of [3H]thymidine incorporation (an index of proliferation) and on adrenomedullin-stimulated nucleosome-associated cytoplasmic DNA fragmentation (an index of apoptosis) in mesangial cells, using a selective inhibitor of P38 MAPK, SB203580 [[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-im idazole], and also to characterize the proximal signal transduction pathways of the three MAPKs in relation to [3H]thymidine incorporation and cytoplasmic DNA fragmentation using a phosphotidyl inositol-3-kinase inhibitor, wortmannin [[1S-(1alpha,6b alpha,9alphabeta,11alpha,11b beta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxyme thyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9- trione]. SB203580 significantly reversed the effects of adrenomedullin on [3H]thymidine incorporation and cytoplasmic DNA fragmentation, and inhibited only P38 MAPK activity. It had no effect on ERK2 and JNK1 activities. Wortmannin, on the other hand, inhibited only adrenomedullin-stimulated cytoplasmic DNA fragmentation and did not affect adrenomedullin-mediated inhibition of [3H]thymidine incorporation. Wortmannin also inhibited adrenomedullin-stimulated P38 MAPK activity without affecting ERK2 and JNK1 activities. These results indicate that: (a) In rat mesangial cells adrenomedullin-mediated inhibition of [3H]thymidine incorporation and stimulation of nucleosome-associated cytoplasmic DNA fragmentation are sensitive to SB203580, and (b) adrenomedullin activates a P38 MAPK through a wortmannin-sensitive kinase. The data using SB203580 suggest an important physiological role for P38 MAPK in rat mesangial cell proliferation and apoptosis.     

活性氧激活的ERK1/2通路在化学性缺氧损伤PC12细胞中的作用

目的探讨胞外信号调节激酶1/2(ERK1/2)在化学性缺氧损伤PC12细胞中的作用。方法应用化学性低氧模拟剂氯化钴(CoCl2)处理PC12细胞建立化学性缺氧损伤模型。应用细胞计数试剂盒-8(cell counting kit-8,CCK-8)比色法检测细胞存活率;罗丹明123(Rh123)染色荧光显微镜照像检测线粒体膜电位(MMP);流式细胞术检测凋亡细胞百分比;Western blot法测定caspase-3、ERK1/2和p38MAPK蛋白的表达水平。结果应用600μmol.L-1处理PC12细胞60 min可使磷酸化(p)ERK1/2的表达明显升高;在600μmol.L-1CoCl2处理PC12细胞前,应用500μmol.L-1N-乙酰半胱氨酸(NAC,为活性氧的清除剂)预处理1 h可抑制CoCl2对p-ERK1/2表达的上调作用;在600μmol.L-1CoCl2处理PC12细胞前,应用10μmol.L-1U0126(ERK1/2抑制剂)预处理2 h可保护PC12细胞对抗CoCl2引起的损伤,使细胞存活率升高,凋亡细胞数目和cleaved caspase-3表达及线粒体膜电位(MMP)丢失均减少;10μmol.L-1U0126预处理还能抑制CoCl2对p-p38MAPK表达的上调作用。此外,应用20μmol.L-1SB203580(p38MAPK抑制剂)预处理能抑制CoCl2对p-ERK1/2表达的上调作用。结论活性氧激活的ERK1/2通路介导CoCl2对PC12细胞的损伤作用,并与p38MAPK通路存在相互的的激活作用。     

Activation and involvement of JNK1 ／ 2 in hydrogen peroxideinduced neurotoxicity in cultured rat cortical neurons

To investigate the role of c-Jun N-terminal protein kinase 1 and 2 (JNK1/2) and the main signal pathway for its activation in hydrogen peroxide (H202) induced apoptotic-like cortical cell death. METHODS: Using the model of oxidative stress induced by I-I202, the expression and diphosphorylation of JNK1/2 was examined by immunoblotting analysis, and neuronal apoptotic like cell death was determined by 4‘,6-diamidino-2-phenylindole (DAPI) staining.RESULTS: The elevation in diphosphorylation level of JNK1/2 (4.40-/5.61-fold vs sham control) was associated with the concentration of H202 (0-100 pmol/L) and the development of apoptotic-like cell death (11.04 %-81.01%).There was no alteration of JNK1/2 protein expression following H202 treatment and recovery at different time points. Administration with JNK1/2 antisense oligonucleotides not only significantly decreased JNK1/2 proteinexpression and activation level, but also significantly reduced cortical cell death induced by H2O2 exposure.Furthermore, both JNKI/2 diphosphorylation and apoptotic-like cell death were largely prevented by pretreat mentwith (5S, 10R)-(-)-5-methyl- 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801)or omission of Ca2 in incubation medium with ethylene glycol-bis(2-aminoethylether)-N,N,N‘,N‘-tetraacefic acid(EGTA). CONCLUSION: JNK1/2 is activated and participates in H202-induced apoptotic-like death in cultured rat cortical neurons mainiv via N-methvi-D-asDartate (NMDA) receptor-mediated influx of extraceUular Ca2 .     

A therapeutic dose of the lipophilic statin pitavastatin enhances oxidant-induced apoptosis in human vascular smooth muscle cells

We examined effects of a physiologic concentration of pitavastatin (0.01 micromol/L) on oxidant-induced apoptosis in cultured human vascular smooth muscle cells (VSMCs). Apoptosis was induced in VSMCs by hydrogen peroxide (H2O2, 300 micromol/L), as evidenced by in situ nick end-labeling and scanning electron microscopy. This apoptotic response was accompanied by increased activation of mitogen-activated protein kinases (MAPKs--ie, increases in the phosphorylated forms of extracellular signal-regulated kinase (p-ERK), c-Jun N-terminal kinase (p-JNK), and p38 MAPK (p-p38 MAPK). Although pitavastatin alone did not induce VSMC death, pretreatment with pitavastatin significantly enhanced H2O2-induced apoptosis and prolonged activation of JNK and p38 MAPK (for up to 24 h) but not ERK. Expression of MAPK phosphatase-1 (MKP-1) also was upregulated by H2O2, but this was not affected by pitavastatin. The apoptosis accelerating effect was observed also in simvastatin but not in pravastatin. Treating VSMCs with mevalonate, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate completely blocked the statin-induced enhancement of VSMC apoptosis, suggesting that protein prenylation is critically involved. It thus appears that pitavastatin enhances H2O2-induced VSMC apoptosis, at least in part, via increases in MAPK activation and protein prenylation, but independently of MKP-1 expression, which consequently results in reduction of VSMC population.     

Role of mitogen-activated protein kinases in hydrogen peroxide-induced cell death in osteoblastic cells

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. However, the underlying mechanism by which oxidants induce cell death remains unclear. The present study was undertaken to determine the role of the mitogen-activated protein kinase subfamilies in hydrogen peroxide (H2O2)-induced cell death of osteoblastic cells. H2O2 resulted in a time- and dose-dependent cell death, which was, in part, attributed to apoptosis. H2O2-induced cell death was prevented by iron chelator, hydroxyl radical scavengers. But H2O2-induced cell death was not affected by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase activation. H2O2 treatment caused a transient activation of extracellular signal-regulated kinase (ERK), followed by sustained activation. Cell death induced by H2O2 was prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2. But H2O2 induced a transient activation of p38 and c-Jun N-terminal kinase (JNK) without sustained activation and inhibitors of these kinses were not effective in preventing the cell death. H2O2 increased Bax expression and produced hyperpolarization of mitochondrial membrane potential and its effect was prevented by PD98059. The ERK activation and cell death induced by H2O2 were not dependent on the phosphorylation of epidermal growth factor receptor. Taken together, these findings suggest that the ERK signaling pathway plays an active role in mediating H2O2-induced apoptosis of osteoblasts and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

Mitogen-activated protein kinase inhibitors differently affect the growth inhibition and death of a proteasome inhibitor, MG132-treated human pulmonary fibroblast cells

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor can induce growth inhibition and death in lung cancer or normal cells. However, little is known about relationship between proteasome inhibition and mitogen-activated protein kinase (MAPK) signaling in normal lung cells. Thus, in the present study, we investigated the effects of MAPK inhibitors on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition, cell death, reactive oxygen species (ROS) and glutathione (GSH). Treatment with 15 μM MG132 increased ROS levels including mitochondrial O(2?)(-) and GSH depleted cell numbers in HPF cells at 24 hours. MAP kinase or ERK kinase (MEK) inhibitor did not significantly affect cell growth inhibition, cell death, the loss of mitochondrial membrane potential (MMP; ΔΨ(m)), ROS level and GSH depletion in MG132-treated HPF cells. c-Jun N-terminal kinase (JNK) inhibitor attenuated the growth inhibition and death by MG132. This inhibitor also significantly decreased O(2?)(-) level in MG132-treated HPF cells. Although p38 inhibitor slightly enhanced HPF cell growth inhibition by MG132, this inhibitor and siRNA prevented HPF cell death induced by MG132. p38 inhibitor also attenuated d O(2?)(-) level and GSH depletion. Moreover, extracellular signal regulated kinase (ERK), JNK or p38 siRNA did not strongly affect ROS levels in MG132-treated HPF cells. ERK and JNK siRNAs decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. In conclusion, MAPK inhibitors differently affected the growth inhibition and death of MG132-treated HPF cells. Especially, p38 inhibitor attenuated HPF cell death by MG132, which was in part related to changes in ROS and GSH levels.     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

Inhibition of UVB-induced oxidative stress-mediated phosphorylation of mitogen-activated protein kinase signaling pathways in cultured human epidermal keratinocytes by green tea polyphenol (-)-epigallocatechin-3-gallate

Exposure of normal human epidermal keratinocytes (NHEK) to UVB radiation induces intracellular release of hydrogen peroxide (oxidative stress) and phosphorylation of mitogen-activated protein kinase cell signaling pathways. Here, we demonstrate that pretreatment of NHEK with (-)-epigallocatechin-3-gallate (EGCG), an antioxidant from green tea, inhibits UVB-induced hydrogen peroxide (H(2)O(2)) production and H(2)O(2)-mediated phosphorylation of MAPK signaling pathways. We found that treatment of EGCG (20 microg/ml of media) to NHEK before UVB (30 mJ/cm(2)) exposure inhibited UVB-induced H(2)O(2) production (66-80%) concomitant with the inhibition of UVB-induced phosphorylation of ERK1/2 (57-80%), JNK (53-83%), and p38 (50-77%) proteins. To demonstrate whether UVB-induced phosphorylation of MAPK occurs via UVB-induced H(2)O(2) (oxidative stress) production, NHEK were treated with the oxidant H(2)O(2). Treatment of H(2)O(2) to NHEK resulted in phosphorylation of ERK1/2, JNK, and p38. Using the same in vitro system, when these cells were pretreated with EGCG or with the known antioxidant ascorbic acid (as positive control), H(2)O(2)-induced phosphorylation of ERK1/2, JNK, and p38 was found to be significantly inhibited. These findings demonstrate that EGCG has the potential to inhibit UVB-induced oxidative stress-mediated phosphorylation of MAPK signaling pathways, suggesting that EGCG could be useful in attenuation of oxidative stress-mediated and MAPK-caused skin disorders in humans.