Hippocalcin increases phospholipase D2 expression through extracellular signal-regulated kinase activation and lysophosphatidic acid potentiates the hippocalcin-induced phospholipase D2 expression

We have previously isolated a 22 kDa protein from a rat brain which was found to be involved in activating phospholipsae D (PLD), and identified the protein as hippocalcin through sequence analysis. Nevertheless, the function of hippocalcin for PLD activation still remains to be resolved. Here, we proposed that hippocalcin was involved in extracellular signal-regulated kinase (ERK)-mediated PLD2 expression. To elucidate a role of hippocalcin, we made hippocalcin transfected NIH3T3 cells and showed that the expression of PLD2 and basal PLD activity were increased in hippocalcin transfected cells. We performed PLD assay with dominant negative PLD2 (DN-PLD2) and hippocalcin co-transfected cells. DN-PLD2 suppressed increase of basal PLD activity in hippocalcin transfected cells, suggesting that increased basal PLD activity is due to PLD2 over-expression. Hippocalcin is a Ca2+-binding protein, which is expressed mainly in the hippocampus. Since it is known that lysophosphatidic acid (LPA) increases intracellular Ca2+, we investigated the possible role of hippocalcin in the LPA-induced elevation of intracellular Ca2+. When the intracellular Ca2+ level was increased by LPA, hippocalcin was translocated to the membrane after LPA treatment in hippocalcin transfected cells. In addition, treatment with LPA in hippocalcin transfected cells markedly potentiated PLD2 expression and showed morphological changes of cell shape suggesting that increased PLD2 expression acts as one of the major factors to cause change of cell shape by making altered membrane lipid composition. Hippocalcin-induced PLD2 expression potentiated by LPA in hippocalcin transfected cells was inhibited by a PI-PLC inhibitor, U73122 and a chelator of intracellular Ca2+, BAPTA-AM suggesting that activation of hippocalcin caused by increased intracellular Ca2+ is important to induce over-expression of PLD2. However, downregulation of PKC and treatment of a chelator of extracellular Ca2+, EGTA had little or no effect on the inhibition of hippocalcin-induced PLD2 expression potentiated by LPA in the hippocalcin transfected cells. Interestingly, when we over-express hippocalcin, ERK was activated, and treatment with LPA in hippocalcin transfected cells significantly potentiated ERK activation. Specific inhibition of ERK dramatically abolished hippocalcin-induced PLD2 expression. Taken together, these results suggest for the first time that hippocalcin can induce PLD2 expression and LPA potentiates hippocalcin-induced PLD2 expression, which is mediated by ERK activation.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

Inhibition of adipocyte differentiation by phytoestrogen genistein through a potential downregulation of extracellular signal-regulated kinases 1/2 activity

In the current study, we investigated the effects of genistein on adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures and its potential signaling pathway. The terminal adipogenic differentiation was assessed by western-blotting analysis of adipogenic-specific proteins such as PPARgamma, C/EBPalpha, and aP2 and the formation of adipocytes. Treatment of mouse BMSC cultures with adipogenic cocktail resulted in sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family, at the early phase of adipogenesis (from days 3 to 9). Inhibition of ERK1/2 activation by PD98059, a specific MEK inhibitor, reversed the induced adipogenic differentiation. Genistein dose-dependently decreased the phosphorylation of ERK1/2 in mouse BMSC cultures. Genistein incubation for the entire culture period, as well as that applied during the early phase of the culture period, significantly inhibited the adipogenic differentiation of mouse BMSC cultures. While genistein was incubated at the late stage (after day 9), no inhibitory effect on adipogenic differentiation was observed. BMSC cultures treated with genistein in the presence of fibroblast growth factor-2 (FGF-2), an activator of the ERK1/2 signaling pathway, expressed normal levels of ERK1/2 activity, and, in so doing, are capable of undergoing adipogenesis. Our results suggest that activation of the ERK1/2 signaling pathway during the early phase of adipogenesis (from days 3 to 9) is essential to adipogenic differentiation of BMSC cultures, and that genistein inhibits the adipogenic differentiation through a potential downregulation of ERK1/2 activity at this early phase of adipogenesis.     

Activation of the extracellular signal-regulated kinase pathway contributes to the behavioral deficit of fragile x-syndrome

Fragile X syndrome (FXS) is a developmental disorder caused by the loss of Fragile X Mental Retardation 1 (FMR1) gene function because of a CGG repeat expansion (> 200 repeats) in the gene. The molecular mechanism(s) linking loss of FMR1 function to the molecular pathology and cognitive/behavioral disability remain unclear. Given the critical role of extracellular signal-regulated kinase (ERK) in synaptic plasticity and neurodevelopment, a number of recent studies have investigated ERK phosphorylation under basal conditions or upon mGluR-induction using neuronal and peripheral tissues from Fmr1 knockout mice and peripheral tissues from FXS patients. However, these reports have presented conflicting results. The current study is the first to focus on the levels of ERK phosphorylation in brain tissue from human FXS patients. In both human brain tissue and brain tissue from Fmr1 knockout mice there was significantly increased phosphorylation of MEK1/2 and ERK. Indeed, treating Fmr1 knockout mice with the MEK1/2 inhibitor SL327 abrogated audiogenic seizure activity, a feature of the Fmr1 knockout mice that replicates the symptom in patients with FXS. These findings suggest that activation of the ERK pathway results in some cardinal cognitive and clinical features in FXS patients and likely have profound translational implications.     

Resveratrol reduces oxidation and proliferation of human retinal pigment epithelial cells via extracellular signal-regulated kinase inhibition

Epidemiological evidence suggests that moderate wine consumption and antioxidant-rich diets may protect against age-related macular degeneration (AMD), the leading cause of vision loss among the elderly. Development of AMD and other retinal diseases, such as proliferative vitreoretinopathy (PVR), is associated with oxidative stress in the retinal pigment epithelium (RPE), a cell layer responsible for maintaining the health of the retina by providing structural and nutritional support. We hypothesize that resveratrol, a red wine polyphenol, may be responsible, in part, for the health benefits of moderate red wine consumption on retinal disease. To test this hypothesis, the antioxidant and antiproliferative effects of resveratrol were examined in a human RPE cell line (designated ARPE-19). Cell proliferation was determined using the bromodeoxyuridine (BrdU) assay, intracellular oxidation was assessed by dichlorofluorescein fluorescence, and activation of the mitogen-activated protein kinase (MAPK) cascade was measured by immunoblotting. Treatment with 50 and 100 micromol/L resveratrol significantly reduced proliferation of RPE cells by 10% and 25%, respectively (P<0.05). This reduction in proliferation was not associated with resveratrol-induced cytotoxicity. Resveratrol (100 micromol/L) inhibited basal and H2O2-induced intracellular oxidation and protected RPE cells from H2O2-induced cell death. The observed reduction in cell proliferation was associated with inhibition of mitogen activated protein kinase/ERK (MEK) and extracellular signal-regulated kinase (ERK 1/2) activities at concentrations of resveratrol as low as 5 micromol/L. These results suggest that resveratrol can reduce oxidative stress and hyperproliferation of the RPE.     

Luteolin induces apoptotic cell death through AIF nuclear translocation mediated by activation of ERK and p38 in human breast cancer cell lines

The flavonoid, luteolin, has been shown to have anticancer activity in various cancer cells; however, the precise molecular mechanism of its action is not completely understood, and studies were conducted to find out how it induces apoptosis in breast cancer cells. Luteolin induced a reduction of viability in a dose- and time-dependent manner. The pro-apoptotic effect of luteolin was demonstrated by cell cycle measurement and Hoechst 3325 staining. Western blot analysis showed that luteolin activates ERK (extracellular-signal-regulated kinase) and p38. Pharmacological inhibition or knockdown of ERK and p38 protected against luteolin-induced cell death; however, the caspase-3-specific inhibitor had no effect. Immunocytochemical examination indicated that luteolin induced nuclear translocation of AIF (apoptosis-inducing factor), which was mediated by activation of ERK and p38. Transfection of a vector expressing the miRNA (microRNA) of AIF prevented luteolin-induced apoptosis. The data suggest that luteolin induces a caspase-dependent and -independent apoptosis involving AIF nuclear translocation mediated by activation of ERK and p38 in breast cancer cells.     

Acute physical exercise increases leptin-induced hypothalamic extracellular signal-regulated kinase1/2 phosphorylation and thermogenesis of obese mice

The obesity is a result of energy imbalance and the increase in thermogenesis seems an interesting alternative for the treatment of this disease. The mechanism of energy expenditure through thermogenesis is tightly articulated in the hypothalamus by leptin. The hypothalamic extracellular signal-regulated kinase-1/2 (ERK1/2) is a key mediator of the thermoregulatory effect of leptin and mediates the sympathetic signal to the brown adipose tissue (BAT). In this context, physical exercise is one of the main interventions for the treatment of obesity. Thus, this study aimed to verify the effects of acute physical exercise on leptin-induced hypothalamic ERK1/2 phosphorylation and thermogenesis in obese mice. Here we showed that acute physical exercise reduced the fasting glucose of obese mice and increased leptin-induced hypothalamic p-ERK1/2 and uncoupling protein 1 (UCP1) content in BAT ( P < 0.05). These molecular changes are accompanied by an increased oxygen uptake (VO ₂) and heat production in obese exercised mice ( P < 0.05). The increased energy expenditure in the obese exercised animals occurred independently of changes in spontaneous activity. Thus, this is the first study demonstrating that acute physical exercise can increase leptin-induced hypothalamic ERK1/2 phosphorylation and energy expenditure of obese mice.     

Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1)

Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.     

Neuroprotection of immortalized hippocampal neurones by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase

We have investigated the molecular mechanisms of neurotrophin-mediated cell survival in HT22 cells, a murine cell line of hippocampal origin, expressing the brain-derived neurotrophic factor (BDNF) receptor TrkB as well as the TrkB.T1 splice variant. Stimulation with BDNF protected HT22-TrkB cells, but not HT22-TrkB.T1 cells, against programmed cell death induced by serum deprivation. BDNF did not, however, provide protection against oxidative glutamate toxicity, indicating that serum deprivation-induced cell death differs substantially from glutamate-induced cell death. Using a pharmacological strategy to block either the extracellular signal-regulated protein kinase (ERK) or the phosphatidylinositol 3-kinase (PI3) pathway, we show that activation of PI3 kinase is required for the neuroprotective activity of BDNF in HT22 cells. To further analyse the role of ERK in neuroprotection we expressed an inducible deltaRaf-1:ER fusion protein in HT22 cells. Activation of this conditionally active form of Raf-1 induced a sustained phosphorylation of ERK, and protected the cells from serum withdrawal-induced cell death. Inhibition of ERK activation at different time points revealed that a prolonged activation of ERK is essential to protect HT22 cells from cell death triggered by the withdrawal of serum, indicating that the duration of ERK activation is of major importance for its neuroprotective biological function.     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

Histone deacetylase 1 and 2 differentially regulate apoptosis by opposing effects on extracellular signal-regulated kinase 1/2

Histone deacetylases (HDACs) are epigenetic regulators that are important for the control of various pathophysiological events. We found that HDAC inhibitors completely abolished transforming growth factor-β1 (TGF-β1)-induced apoptosis in AML-12 and primary mouse hepatocytes. Expression of a dominant-negative mutant of HDAC1 or downregulation of HDAC1 by RNAi both suppressed TGF-β1-induced apoptosis. In addition, overexpression of HDAC1 enhanced TGF-β1-induced apoptosis, and the rescue of HDAC1 expression in HDAC1 RNAi cells restored the apoptotic response of cells to TGF-β1. These data indicate that HDAC1 functions as a proapoptotic factor in TGF-β1-induced apoptosis. In contrast, downregulation of HDAC2 by RNAi increased spontaneous apoptosis and markedly enhanced TGF-β1-induced apoptosis, suggesting that HDAC2 has a reciprocal role in controlling cell survival. Furthermore, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) by MEK1 inhibitor PD98059 or expression of a kinase-dead mutant of MEK1 restored the apoptotic response to TGF-β1 in HDAC1 RNAi cells. Strikingly, HDAC2 RNAi caused an inhibition of ERK1/2, and the spontaneous apoptosis can be abolished by reactivation of ERK1/2. Taken together, our data demonstrate that HDAC1 and 2 reciprocally affect cell viability by differential regulation of ERK1/2; these observations provide insight into the roles and potential mechanisms of HDAC1 and 2 in apoptosis.     

Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities but its implementation has been slow. One of the reasons is the lack of full understanding of the complexities of cellular pathways through which signals for differentiation lead to cell maturation. This was addressed in this study using HL60 cells, a well-established model of differentiation of neoplastic cells. SB 203580 and SB 202190, specific inhibitors of a signaling protein p38 MAP kinase, were found to markedly accelerate monocytic differentiation of HL60 cells induced by low concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)). Surprisingly, inhibition of p38 activity resulted in sustained enhancement of p38 phosphorylation and of its in vitro activity in the absence of the inhibitor, indicating up-regulation of the upstream components of the p38 pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted in a prolonged activation of the JNK and, to a lesser extent, the ERK pathways. The data are consistent with the hypothesis that in HL60 cells an interruption of a negative feedback loop from a p38 target activates a common regulator of multiple MAPK pathways. The possibility also exists that JNK and/or ERK pathways amplify a differentiation signal provided by 1,25D(3).     

Deregulation of cyclin-dependent kinase 2 activity correlates with UVC-induced apoptosis in Chinese hamster ovary cells

Progression through the cell cycle relies on the activities of cyclin-dependent kinases (Cdk), which in turn are modulated by inhibitory proteins such as p21(waf1/cip1) that are induced when genomic damage occurs. In this study, we show that exposure of normal mammalian cells, such as NIH3T3 fibroblasts, to UVC (25 J/m2, at 254 nm) induces the expression of p21 without causing significant apoptosis, whereas similar treatment of Chinese hamster ovary (CHO-K1) cells with UVC causes apoptosis without inducing p21. The absence of p21 in UV-irradiated CHO-K1 cells is accompanied by the deregulation of Cdk2 activity. The elevation of Cdk2 activity correlates with the increase of UV-induced apoptosis, which can be suppressed by small-molecule Cdk2 inhibitors such as roscovitine and pyrrolidine dithiocarbamate. The results of this study suggest that the deregulation of Cdk2 activity may be critical to UV-induced apoptosis in CHO-K1 cells.     

Control of FLIPL expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells

Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP_L) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP_L levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP_L upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP_L expression. FLIP_L upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP_L upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP_L levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated.     

Inhibition of the extracellular signal-regulated kinase signaling pathway is correlated with proteasome inhibitor suppression of coxsackievirus replication

The ubiquitin/proteasome system (UPS), a major intracellular protein degradation pathway, plays a critical role in coxsackieviral replication. To elucidate the mechanisms by which the UPS regulates viral replication, we studied the influence of proteasome inhibition on signaling through the extracellular signal-regulated kinase (ERK) pathway, a pathway which has been previously demonstrated to be necessary for coxsackieviral replication and contribute to virus-mediated pathogenesis. We found that proteasome inhibition reduced coxsackievirus-induced ERK phosphorylation in a dose-dependent manner, which is correlated with an induction of the mitogen-activated protein kinase phosphatase-1 (MKP-1). Blockade of MKP induction by short-interfering RNA attenuated the loss of ERK phosphorylation, and subsequently restored viral replication. Our results suggest that inhibition of the ERK signaling pathway contributes, as least in part, to proteasome inhibitor-mediated reduction of coxsackievirus replication, demonstrating a converging function of major intracellular signaling and protein degradation pathways in the regulation of viral replication.     

Regulation of matrix Gla protein by parathyroid hormone in MC3T3-E1 osteoblast-like cells involves protein kinase A and extracellular signal-regulated kinase pathways

Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix gamma-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a -748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 (-7) M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co-transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

HaRas activates the NADPH oxidase complex in human neuroblastoma cells via extracellular signal-regulated kinase 1/2 pathway

In this study we have investigated the effects of the small GTP-binding-protein Ras on the redox signalling of the human neuroblastoma cell line, SK-N-BE stably transfected with HaRas(Val12). The levels of reactive oxygen species (ROS) and superoxide anions were significantly higher in HaRas(Val12) expressing (SK-HaRas) cells than in control cells. The treatment of cells with 4-(2-aminoethyl) benzenesulfonylfluoride, a specific inhibitor of the membrane superoxide generating system NADPH oxidase, suppressed the rise in ROS and significantly reduced superoxide levels produced by SK-HaRas cells. Moreover, HaRas(Val12) induced the translocation of the cytosolic components of the NADPH oxidase complex p67(phox) and Rac to the plasma membrane. These effects depended on the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK1/2) pathway, as the specific MEK inhibitor, PD98059, prevented HaRas-mediated increase in ROS and superoxide anions. In contrast, the specific phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and wortmannin were unable to reverse the effects of HaRas(Val12). Moreover, cholinergic stimulation of neuroblastoma cells by carbachol, which activated endogenous Ras/ERK1/2, induced a significant increase in ROS levels and elicited membrane translocation of p67(phox) and Rac. ROS generation induced by carbachol required the activation of ERK1/2 and PI3K. Hence, these data indicate that HaRas-induced ERK1/2 signalling selectively activates NADPH oxidase system in neuroblastoma cells.