Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS). This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H(2)O(2), because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H(2)O(2), acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.     

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.     

M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase- and MAPK/ERK-independent pathways

When PC12 cells are deprived of trophic support they undergo apoptosis. We have previously shown that survival of trophic factor-deprived PC12M1 cells can be promoted by activation of the G protein-coupled muscarinic receptors. The mechanism whereby muscarinic receptors inhibit apoptosis is poorly understood. In the present study we investigated this mechanism by examining the effect of muscarinic receptor activation on the serum deprivation-induced activity of key players in apoptosis, the caspases, in PC12M1 cells. The results showed that m1 muscarinic activation inhibits caspase activity induced by serum deprivation. This effect appeared to be caused by the prevention of activation of caspases such as caspase-2 and caspase-3, and not by the inhibition of existing activity. Muscarinic receptor activation also stimulated the mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/ERK) and phosphoinositide (PI) 3-kinase signaling pathways. The PI 3-kinase pathway inhibitors wortmannin and LY294002, as well as the MAPK/ERK pathway PD98059 inhibitor, did not however suppress the inhibitory effect of the muscarinic receptors on caspase activity. The results therefore suggested that the muscarinic survival effect is mediated by a pathway that leads to caspase inhibition by MAPK/ERK- and PI 3-kinase-independent signaling cascades.     

Butylene fipronil induces apoptosis in PC12 murine nervous cells via activation of p16‐CDK4/6‐cyclin D1 and mitochondrial apoptotic pathway

Butylene fipronil (BFPN) is a phenylpyrazole insecticide, acting at the γ‐aminobutyric acid (GABA) receptor. Here, we show that BFPN inducedcytotoxicity in PC12 murinenervous cells, which lacks GABA receptor. Treatment with BFPN for 48 hours significantly enhanced G0/G1 arrest and induced apoptosis. BFPN decreased the expression of cyclin‐dependent kinase (CDK4 and CDK6) and increased P16 and cyclin D1. Simultaneously, Bcl‐2 protein was declined while Bax and cytochrome c were significantly enhanced in BFPN‐treated groups. The apoptotic enzymes caspase‐8, ‐9, and ‐3 were also activated by BFPN. Furthermore, treatment with BFPN significantly stimulated reactive oxygen species (ROS) generation, and pretreatment with antioxidant diphenyleneiodonium, substantially reduced cell death. Overall, these results suggest that BFPN is effective to induce G0/G1‐phase arrest and apoptosis in PC12 murine nervous cell. Stimulating ROS generation and activation of P16‐CDK4/6‐cyclin D1 and mitochondrial apoptotic pathway may participate in the cytotoxicity of BFPN.     

Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR.

Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitric-oxide synthase inhibitors diphenyleneiodonium and N(G)methyl-l-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-gamma-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-kappa B pathway

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-kappa B site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and I kappa B alpha partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-kappa B. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of I kappa B beta (but not I kappa B alpha), and increase in NF-kappa B DNA binding activity. Consistent with the idea that ROS may participate in mdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-kappa B DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of I kappa B beta, and subsequent activation of NF-kappa B. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.     

Growth Factor-Like Effects Mediated by Muscarinic Receptors in PC12M1 Cells

Rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors (PC12M1) undergo morphological changes when stimulated by muscarinic agonists. These changes, which include the outgrowth of neurite-like processes, are blocked by the muscarinic antagonist atropine and are not observed in PC12 cells. The observed morphological changes, which are independent of RNA and protein synthesis, are blocked by the methylation inhibitor 5'-deoxy-5'-methylthioadenosine, suggesting that methylation plays a role in this process. Analysis of cyclic AMP accumulation and phosphoinositide turnover reveals that both processes are enhanced on activation by muscarinic agonist. Our data suggest, however, that the muscarinic-dependent neurite-like outgrowth processes are not mediated by cyclic AMP, Ca2+, or protein kinase C pathways. The muscarinic-dependent neurite outgrowth effect is enhanced by nerve growth factor, with a resulting increase in both the number of neurite-extending cells and the length of the neurite. In addition, activation of muscarinic receptors in PC12M1 cells stimulates the induction of marker genes for neuronal differentiation. Muscarinic receptors may therefore mediate growth factor-like effects in these cells.     

Stem cell factor and H2O2 induce GLUT1 translocation in M07e cells

This work aims to elucidate the mechanisms involved in the early activation of glucose transport in hematopoietic M07e cells by stem cell factor (SCF) and a reactive oxygen species (ROS) as H2O2. SCF and H2O2 increase Vmax for glucose transport; this enhancement is due to a higher content in GLUT1 in plasma membranes, possibly through a translocation from intracellular stores. Inhibitors of tyrosine kinases or phospholipase C (PLC) remove glucose transport enhancement and prevent translocation. The inhibitory effect of STI-571 suggests a role for c-kit tyrosine kinase on glucose transport activation not only by SCF, but also by H2O2. On the other hand, neither protein kinase C nor phosphoinositide-3-kinase appear to be involved in the acute activation of glucose transport. Our data suggest that i) in M07e cells, SCF and exogenous H2O2 elicit a short-term activation of glucose transport through a translocation of GLUT1 from intracellular stores to plasma membranes; ii) both stimuli could share at least some signaling pathways leading to glucose uptake activation, involving protein tyrosine kinases and PLC iii) H2O2 could act increasing the level of tyrosine phosphorylation through the inhibition of tyrosine phosphatases and mimicking the regulation role of endogenous ROS.     

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells

Abstract

Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H₂O₂ accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.     

Human cytomegalovirus induces multiple means to combat reactive oxygen species

Reactive oxygen species (ROS) are generated as by-products of many cellular processes and can modulate cellular signaling pathways. However, high ROS levels are toxic; thus, intracellular ROS need to be tightly controlled. Therefore, cells use a group of antioxidant molecules and detoxifying enzymes that remove or detoxify reactive species. We found that the level of the antioxidant glutathione is greatly increased in human cytomegalovirus (HCMV)-infected cells due to activation of glutathione synthetic enzymes. In addition, our data suggest that virus-specific mechanisms are used to induce the expression of target antioxidant and detoxifying enzymes critical for the success of the infection. As a result of this virus-induced anti-ROS environment, key signaling kinases, such as the mammalian target of rapamycin (mTOR) kinase in mTOR complex 1 (mTORC1), are protected from inhibition by exogenous hydrogen peroxide (H(2)O(2)). In this regard, we found that phosphorylation of mTOR kinase at serine 2448 (suggested to be activating) was maintained during infection even under ROS stress conditions that inhibited it in uninfected cells. We also show that AMP-dependent kinase (AMPK)-mediated phosphorylation of serine 792 of raptor, the specificity subunit of mTORC1, increases in infected cells after H(2)O(2) treatment. This phosphorylation is normally inhibitory for mTORC1. However, in infected cells this did not result in inhibition of mTORC1 activity, suggesting that inhibitory effects of raptor phosphorylation are circumvented. Overall, our data suggest that HCMV utilizes virus-specific mechanisms to activate a variety of means to protect the cell and mTORC1 from the effects of ROS.     

Calcium signaling is involved in cadmium-induced neuronal apoptosis via induction of reactive oxygen species and activation of MAPK/mTOR network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+](i)) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+](i) elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+](i) elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+](i) elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+](i), which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+](i) homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.     

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.     

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by reactive oxygen species

Intrinsic expression of the multidrug resistance (MDR) transporter P-glycoprotein (Pgp) may be regulated by reactive oxygen species (ROS). A transient expression of Pgp was observed during the growth of multicellular tumor spheroids. Maximum Pgp expression occurred in tumor spheroids with a high percentage of quiescent, Ki-67-negative cells, elevated glutathione levels, increased expression of the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF-1 as well as reduced ROS levels and minor activity of the mitogen-activated kinase (MAPK) members c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase ERK1,2, and p38 MAPK. Raising intracellular ROS by depletion of glutathione with buthionine sulfoximine (BSO) or glutamine starvation resulted in down-regulation of Pgp and p27Kip1, whereas ERK1,2 and JNK were activated. Down-regulation of Pgp was furthermore observed with low concentrations of hydrogen peroxide and epidermal growth factor, indicating that ROS may regulate Pgp expression. The down-regulation of Pgp following BSO treatment was abolished by agents interfering with receptor tyrosine kinase signaling pathways, i.e. the protein kinase C inhibitors bisindolylmaleimide I (BIM-1) and Ro-31-8220, the p21ras farnesyl protein transferase inhibitor III, the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1,2 activation. ROS involved as second messengers in receptor tyrosine kinase signaling pathways may act as negative regulators of Pgp expression.