Nerve growth factor protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a phosphatidylinositol 3-kinase-dependent manner

The survival signal elicited by the phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated with inactivation of pro-apoptotic proteins and attenuation of the general stress-induced increase in reactive oxygen species (ROS). However, the mechanisms by which this pathway regulates intracellular ROS levels remain largely unexplored. In this study, we demonstrate that nerve growth factor (NGF) prevents the accumulation of ROS in dopaminergic PC12 cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves PI3K/Akt-dependent induction of the stress response protein heme oxygenase-1 (HO-1). The effect of NGF was mimicked by induction of HO-1 expression with CoCl(2); by treatment with bilirubin, an end product of heme catabolism; and by infection with a retroviral expression vector for human HO-1. The relevance of HO-1 in NGF-induced ROS reduction was further demonstrated by the evidence that cells treated with the HO-1 inhibitor tin-protoporphyrin or infected with a retroviral expression vector for antisense HO-1 exhibited enhanced ROS release in response to 6-OHDA, despite the presence of the neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1 mRNA and protein and partially reversed its protective effect against 6-OHDA-induced ROS release. By contrast, cells transfected with a membrane-targeted active version of Akt1 exhibited increased HO-1 expression, even in the absence of NGF, and displayed a greatly attenuated production of ROS and apoptosis in response to 6-OHDA. These observations indicate that the PI3K/Akt pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

Bcl-x(S) induces an NGF-inhibitable cytochrome c release

Bcl-x(S), a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-x(S) and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-x(S) induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-x(S) induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-x(S)-induced apoptosis, and that NGF inhibits Bcl-x(S)-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways.     

Effects of scutellarin on apoptosis induced by cobalt chloride in PC12 cells

The present study investigated the protective effects of scutellarin on cobalt chloride (CoCl2)-induced apoptosis in PC12 cells. Incubation of PC12 cells with 500 microM CoCl2 for 24 h resulted in significant apoptosis as evaluated by the crystal violet, electron microscopy and flow cytometry assays. The increase of caspase-3 activity, decrease of Bcl-XL expression, phosphorylation of p38 mitogen-activated protein kinase (MAPK) and accumulation of intracellular reactive oxygen species (ROS) were also seen in CoCl2-treated PC12 cells. Scutellarin at 0.1, 1 and 10 microM significantly protected against the apoptotic cell death induced by CoCl2. Scutellarin decreased caspase-3 activity, increased Bcl-XL expression, inhibited p38 phosphorylation and attenuated ROS production. These results demonstrate that scutellarin can protect PC12 cells from cobalt chloride induced apoptosis by scavenging ROS, inhibiting p38 phosphorylation, up-regulating Bcl-XL expression and decreasing caspase-3 activity, and may reduce the cellular damage in pathological conditions associated with hypoxia-mediated neuronal injury.     

Differential effect of nerve growth factor on dopaminergic neurotoxin-induced apoptosis

Both rotenone and manganese are possible neurotoxins for a wide variety of cell and neuronal types including dopaminergic neurons and induce apoptosis in various cells. Neurotrophic factors have the potential for therapeutic development when used to prevent Parkinson's disease. In this paper, we focused on the differences between rotenone and manganese as toxins, and characterized the influence of neurotrophic factors on toxin-induced apoptosis in PC12 cells. There were distinct differences in intracellular mechanisms between rotenone- and manganese-induced apoptosis such as the production of reactive oxygen species, the response to antioxidants, and the activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Nerve growth factor (NGF) almost completely prevented rotenone-induced but not manganese-induced caspase activation and DNA fragmentation. The differential effect of NGF was found to be mainly due to the down-regulation of the Trk tyrosine kinase receptor by manganese but not by rotenone. Prevention of rotenone-induced apoptosis by NGF was attenuated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, but not MAPK kinase (MEK) inhibitors, PD98059 or U0126. These results demonstrate that the potential neurotoxins for dopaminergic cells exert their toxic effect by activation of different signaling pathways of apoptosis and that NGF prevents rotenone-induced apoptosis through the activation of the PI 3-kinase pathway not MAPK pathway.     

Gastrodia elata prevents rat pheochromocytoma cells from serum-deprived apoptosis: the role of the MAPK family

Gastrodia elata (G. elata) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, and epilepsy. In this study, differential methanol (MeOH) extracts of G. elata were found to prevent serum-deprived rat pheochromocytoma (PC12) cell apoptosis by the MTT assay and Hoechst staining. A serine/threonine kinase inhibitor attenuated this protection. G. elata resulted in phosphorylation and dephosphorylation of ERK1/2 and JNK1/2-p38 MAPKs (members of the serine/threonine kinase family), respectively, as revealed by Western blot analysis. An upstream ERK inhibitor attenuated G. elata-induced ERK phosphorylation but not protective effect. Although JNK and p38 inhibitors attenuated their related enzyme activities during serum deprivation, only JNK inhibitor prevented serum-deprived apoptosis. Thus, G. elata prevents serum-deprived apoptosis through activation of the serine/threonine kinase-dependent pathway and suppression of JNK activity.     

Carbon monoxide protects PC12 cells from peroxynitrite-induced apoptotic death by preventing the depolarization of mitochondrial transmembrane potential

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in catalyzing heme degradation into biliverdin, free iron, and carbon monoxide (CO), serves as a protective enzyme against oxidative and nitrosative stresses. In the present study, we investigated the cytoprotective effects of HO-1 upregulation and its product CO against the peroxynitrite-induced PC12 cell death. PC12 cells treated with 3-morphoinosydonimine (SIN-1), a generator of peroxynitrite (ONOO-), underwent apoptotic cell death as evidenced by dissipation of mitochondrial transmembrane potential (DeltaPsim), release of mitochondrial cytochrome c into cytoplasm, cleavage of poly(ADP-ribose)polymerase and fragmentation of internucleosomal DNA. Pretreatment of PC12 cells with a low non-toxic concentration of SIN-1 (0.5 mM) induced HO-1 expression and abrogated the cell death caused by subsequent challenge with high dose SIN-1 (2.5 mM). Furthermore, pretreatment of PC12 cells with SnCl2, a potent inducer of HO-1 expression, increased endogenous production of CO (HO activity) and rescued the PC12 cells from peroxynitrite-induced apoptosis. The cytoprotective effect of SnCl2 was abolished when the HO activity was inhibited by zinc protoporphyrin IX (ZnPP IX). PC12 cells treated directly with the CO-releasing molecule, tricarbonyldichlororuthenium (II) dimer ([Ru(CO)3Cl2]2) became tolerant to the depolarization of DeltaPsim and apoptosis induced by high dose peroxynitrite. Taken together, these data demonstrate that the adaptive protection against peroxynitrite-induced apoptotic death in PC12 cells is mediated by CO formed as a consequence of HO-1 induction.     

Multiple G<Subscript>i</Subscript> Proteins Participate in Nerve Growth Factor-Induced Activation of c-Jun N-terminal Kinases in PC12 Cells

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G_i/o proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Gα_z or the PTX-resistant mutants of Gα_i1–3 and Gα_oA. Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G_i1–3 proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.     

Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells

We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF. Rsk1 and Rsk2 mutated in the PDK1-binding site were not activated by either NGF or MEK1-DD. Expression of constitutively active Rsk1 or Rsk2 in PC12 cells resulted in highly active proteins whose levels of activity did not change either with NGF treatment or after coexpression with MEK1-DD. Rsk2-CA expression had no detectable effect on the cells. However, expression of Rsk1-CA led to differentiation of PC12 cells even in the absence of NGF, as evidenced by neurite outgrowth. Differentiation was not observed with a nonactive Rsk1-CA that was mutated in the PDK1-binding site. Expression of Rsk1-CA did not lead to activation of the endogenous MAPK pathway, indicating that Rsk1 is sufficient to induce neurite outgrowth and is the only target of MAPK required for this effect. Collectively, our data demonstrate a key role for Rsk1 in the differentiation process of PC12 cells.     

Piperine protects cisplatin-induced apoptosis via heme oxygenase-1 induction in auditory cells

Piperine is a major component of black pepper, Piper nigrum Linn, used widely in traditional medicine. In this study, we examined whether piperine could protect House Ear Institute-Organ of Corti 1 (HEI-OC1) cells against cisplatin-induced apoptosis through the induction of heme oxygenase (HO)-1 expression. Piperine (10-100 microM) induced the expression of HO-1 in dose- and time-dependent manners. Piperine also induced antioxidant response element-luciferase and translocated nuclear factor-E2-related factor-2 (Nrf2) to nucleus. Piperine activated the c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK) pathways, and the JNK pathway played an important role in piperine-induced HO-1 expression. Piperine protected the cells against cisplatin-induced apoptosis. The protective effect of piperine was abrogated by zinc protoporphyrin IX, an HO inhibitor, and antisense oligodeoxynucleotides against HO-1 gene. These results demonstrate that the expression of HO-1 by piperine is mediated by both JNK pathway and Nrf2, and the expression inhibits cisplatin-induced apoptosis in HEI-OC1 cells.     

Nerve growth factor-induced expression of the GTP cyclohydrolase I gene via Ras/MEK pathway in PC12D cells

Neurotrophins are essential for the development and survival of the catecholaminergic neurons. GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme in the biosynthesis of 5,6,7,8-tertahydrobiopterin (BH4), the required cofactor for tyrosine hydroxylase. Previously, we reported that TH requires the Ras/mitogen-activated protein kinase kinase (MEK) pathway for its induction by nerve growth factor (NGF). Here, we examined intracellular signals required for NGF-induced expression of the GCH gene in PC12D cells. The activity of GCH was increased up to 5-fold after the NGF treatment, and the increase was repressed by pretreatment with U0126, an MEK1/2 inhibitor, but not with protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) inhibitors. Induction of GCH mRNA by NGF was also abolished by pretreatment with U0126. The human GCH promoter activity was significantly enhanced by NGF treatment. Deletion analysis showed that the 465-bp 5'-flanking region is responsible for NGF-enhanced promoter activity. These data suggest that the Ras-MEK pathway is required for coordinate expression of the GCH and TH genes induced by neurotrophins.