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PubMed1997

Induction of neurite outgrowth in PC12 cells by gamma-lactam-related compounds via Ras-MAP kinase signaling pathway independent mechanism.

Rat pheochromocytoma cells, PC12 cells, undergo differentiation in response to nerve growth factor (NGF). Although the Ras-MAP kinase signaling pathway has been shown to play a central role in the response to NGF, the precise mechanism which induces differentiation remains unclarified. Recently, several gamma-lactam-related microbial products were identified to induce neurite outgrowth in neuroblastoma cells. Therefore, we synthesized a series of gamma-lactam-related compounds and tested for their ability to induce neurite outgrowth in PC12 cells. We found that two compounds, MT-19 and MT-20, induced neurite outgrowth at concentrations as low as 1 microg/ml. MT-19 and MT-20 have an n-hexadecyl group and an n-dodecyl group, respectively, at the position N-1 of the gamma-lactam ring, and the modification of this group leads to partial or complete loss of activity. In addition, the modification of the methyl and hydroxyl group at C-5 leads to complete loss of activity, indicating a strict structure-activity relationship. Interestingly, MT-19 and MT-20 induced neurite outgrowth of PC12 cells which lack normal Ras function. Furthermore, these compounds did not induce MAP kinase activation, suggesting that MT-19 and MT-20 do not require the Ras-MAP kinase signaling pathway which is shown to be necessary and sufficient for NGF-induced neurite outgrowth. Consistent with this, none of the early- or late-response genes tested, which include fos, zif268, Nur77, vgf, and transin, was induced. However, the protein level of three neurofilaments was increased after the incubation with these compounds. Since the level of other cytoskeleton proteins including actin and tubulin remained constant, MT-19 and MT-20 specifically affected neurofilament synthesis and/or turnover. Taken together, these findings indicate that MT-19 and MT-20 induce neurite outgrowth by activating the downstream target of MAP kinase or by a novel mechanism which is distinct from the NGF-activated pathway.

Actins↗
PubMed1999

Laminin stimulates protein tyrosine dephosphorylation in PC12 cells.

Laminin stimulates neurite outgrowth in rat pheochromocytoma cells (PC12 cells). Here, we investigated laminin signal transduction mechanisms by adding the tyrosine kinase/phosphatase modulators, genistein, quercetin, aurin tricarboxylic acid (ATA), and vanadate to PC12 cells. At 10 microM both genistein and quercetin enhanced laminin-mediated neurite outgrowth by 1.7- and 2.3-fold, respectively, while at 10 microM, ATA inhibited laminin-mediated neurite outgrowth by 92%. Vanadate inhibited neurite outgrowth by 63% at 10 microM. Immunoblot analysis revealed four proteins of approximately 240, 22, 110, and 35 kDa, which were dephosphorylated on tyrosine residues in laminin-treated PC12 cells, but not in NIH 3T3 cells. These results demonstrate that laminin-mediated neurite outgrowth involves protein tyrosine dephosphorylation and suggests that this mechanism may have specificity to neuronal cells.

Animals↗
PubMed2005

Differential effect of calmodulin antagonists on MG132-induced mitochondrial dysfunction and cell death in PC12 cells.

Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.

Animals↗
PubMed2004

Ca2+ store-independent augmentation of [Ca2+]i responses to G-protein coupled receptor activation in recombinantly TRPC5-expressed rat pheochromocytoma (PC12) cells.

Mammalian homologues of the Drosophila canonical transient receptor potential (trp) protein (TRPC) have been implicated to function as receptor-operated Ca(2+) channels (ROCs) or store-operated Ca(2+) channels (SOCs). To determine the role of TRPC5 protein in neural cells, TRPC5 was recombinantly expressed in rat pheochromocytoma cells (PC12) and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and Na(+) concentration ([Na(+)](i)) were analyzed. TRPC1 and TRPC3 mRNAs were endogenously expressed in PC12 cells. In TRPC5-expressed cells (TRPC5-cells), the resting [Ca(2+)](i) and [Na(+)](i) were significantly higher than those in control cells. The [Ca(2+)](i) increases induced by bradykinin and uridine 5'-triphosphate were significantly larger in TRPC5-cells. TRPC5 expression did not change in store-operated Ca(2+) entry elicited by thapsigarigin. TRPC5-cells showed larger inward current and increase of [Na(+)](i) in response to BK than control cells. These results suggest that TRPC5 channels expressed in PC12 cells function as ROCs activated by G-protein/phospholipase C coupled receptors, but not as SOCs.

Animals↗
PubMed2004

c-FLIP-L recombinant adeno-associated virus vector infection prevents Fas-mediated but not nerve growth factor withdrawal-mediated cell death in PC12 cells.

Fas is a cell surface death receptor that may play an important role in regulating cell death in neuronal cell types by activation of caspase 8. Cellular FLICE inhibitory protein-long (c-FLIP-L) is an endogenous inhibitor of the activation of caspase 8 by Fas. The current study addresses the role of c-FLIP-L in regulation of cell death in PC12 cells induced by nerve growth factor (NGF) withdrawal and Fas antibody, which acts as a Fas ligand and activates the Fas receptor. A recombinant adeno-associated virus (rAAV) vector that expresses c-FLIP-L was constructed. PC12 cells infected with the c-FLIP-L rAAV were resistant to apoptosis induced by treatment with Fas antibody compared to cells infected with enhanced green fluorescent protein (EGFP) expressing rAAV. Overexpression of c-FLIP-L rAAV inhibited cleavage of caspase 8 induced by Fas antibody treatment. In contrast, treatment with the c-FLIP-L rAAV did not protect PC12 cells from cell death induced by NGF withdrawal. In conclusion, overexpression of c-FLIP-L rAAV inhibits Fas antibody-mediated cell death, but not NGF withdrawal-mediated cell death in PC12 cells.

Adenoviridae Infections↗
PubMed1998

A possible role for protein kinase C in CO2/H+-induced c-fos mRNA expression in PC12 cells.

Recently we have found that hypercapnia induces nuclear protein (FOS) expression in the brainstem chemosensitive neurons, including catecholamine-containing cells. In the present studies we examined the role of protein kinase C (PKC) pathway in CO2-induced c-fos expression. Because of the complexity of the CNS system, experiments were performed in pheochromocytoma cells (PC12 cells). These cells originate from neuronal crest and express catecholaminergic traits. We depleted PKC from PC12 cells by prolonged (48 h) exposure to high concentration of phorbol 12-myristate, 13-acetate (PMA, 100 nM), and then determined the expression of: (1) c-fos mRNA by Northern blot (2) PKC isoforms, tyrosine phosphorylated and unphosphorylated MAP (mitogen activated protein) kinases by Western blot. Depletion of PKC abolished the effect of CO2 on c-fos mRNA expression, inhibited MAP kinases tyrosine phosphorylation and suppressed the expression of PKC(alpha) and PKC(zeta). These results suggest that MAP kinases, PKC(alpha) and/or PKC(beta) might be involved in CO2-induced c-fos mRNA expression.

Animals↗
PubMed2000

Priming of PC12 cells for semiquantitative microinjection studies involving Ras.

Nerve growth factor and activated Ras can induce differentiation of rat pheochromocytoma cells (PC12 cells) [Greene and Tischler (1976) Proc. Natl. Acad. Sci. USA 73, 2424-2428] from a chromaffin cell-like morphology into one that resembles sympathetic neurones. We developed a special treatment of PC12 cells which apparently synchronizes these cells such that they are more useful for semi-quantitative microinjection studies for signal transduction pathways. This treatment leads to a faster and more reproducible differentiation which faithfully reproduces the involvement of Ras in the process and allows a comparison of the biological activity of different Ras mutants. It shows that G12V and Q61L oncogenic mutants are not equally potent in inducing differentiation. Partial loss-of-function mutations T35S, E37G and Y40C are inactive and even a triple combination of these does not restore full biological activity.

Amino Acid Substitution↗
PubMed2006

Neuregulin-1 isoform induces mitogenesis, KCa and Ca2+ currents in PC12 cells. A comparison with sciatic nerve conditioned medium.

Neuregulin-1 (NRG-1) is an active component found in sciatic nerve conditioned medium (CM). NRG-1 is a growth and differentiation factor shown to have an effect on neuritogenesis and survival of neural cells. PC12 cells chronically treated with NRG-1 (beta1 isoform) show an increase in proliferation under low-serum condition (2.5% fetal bovine serum and 1.25% horse serum) and serum deprivation, without visible morphological changes. NRG-1 and CM treatments of PC12 cells induced an increase of voltage-activated Ca2+ currents and large-conductance calcium-activated K+ currents (KCa). AG825, a specific inhibitor for erbB2 receptor, abolishes KCa current, though Ca2+ currents were not inhibited. These results showed that NRG-1 is capable of inducing functional changes but is not sufficient on its own to have an effect on cell morphology.

Analysis of Variance↗
PubMed2002

Dual effect of nerve growth factor on cell death of PC12 cells Induced by serum deprivation.

The effect of nerve growth factor (NGF) on the cell death of PC12 cells that is induced by serum deprivation was examined in the floating and attached cells to the extracellular matrix. NGF suppressed cell death occurred in the floating cells. The onset of cell death in the attached cells was much slower than in the floating cells. Moreover, the cell death in the attached cells was either accelerated in a high-density culture (over approximately 50% confluent), or inhibited in a low-density culture by NGF. While nucleosomal DNA fragmentation and poly (ADP-ribose) polymerase degradation was observed in both the floating and attached cells, the incidence of nuclear fragmentation and chromatin condensation was much lower in the attached cells than in the floating cells. The delayed onset of cell death in the attached cells was due to the signals that are generated from the extracellular matrix that is formed by PC12 cells, together with cell-to-cell interaction. The acceleration of cell death in the NGF-treated cells was anoikis, caused by the loss of the anchorage of the cell via the action of increased activities of matrix metalloproteinases (MMP2, MMP9). These results suggest that NGF has a different role in the cell death of PC12 cells that is induced by serum deprivation, depending on the cell-matrix, as well as the cell-cell interaction.

Animals↗
PubMed2006

Protocatechuic acid suppresses MPP+ -induced mitochondrial dysfunction and apoptotic cell death in PC12 cells.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, showed antioxidant neuroprotective effect in our previous study. Here, we investigated the effect of PCA on the MPP(+)-induced mitochondrial dysfunction and apoptotic cell death in PC12 cells. The apoptosis in MPP(+)-induced PC12 cells was associated with loss of mitochondrial membrane potential, the formation of reactive oxygen species (ROS), GSH depletion, activation of caspase-3 and down-regulation of Bcl-2. In contrast, treatment of PC12 cells with PCA significantly prevented the above-mentioned mitochondrial dysfunction. Our data pointed to the potential clinical application/use of PCA to overcome neurodegenerative diseases such as Parkinson's disease.

1-Methyl-4-phenylpyridinium↗
PubMed2001

Involvement of p38 mitogen-activated protein kinase and apoptosis signal-regulating kinase-1 in nitric oxide-induced cell death in PC12 cells.

Although nitric oxide (NO) plays key signaling roles in the nervous systems, excess NO leads to cell death. In this study, the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and apoptosis signal-regulating kinase-1 (ASK1) in NO-induced cell death was investigated in PC12 cells. NO donor transiently activated p38 MAPK in the wild type parental PC12 cells, whereas the p38 MAPK activation was abolished in NO-resistant PC12 cells (PC 12-NO-R). p38 MAPK inhibitors protected the cells against NO-induced death, whereas the inhibitors were not significantly protective against the cytotoxicity of reactive oxygen species. Stable transfection with dominant negative p38 MAPK mutant reduced NO-induced cell death. Stable transfection with dominant negative mutant of ASK1 attenuated NO-stimulated activation of p38 MAPK and decreased NO-induced cell death. These results suggest that p38 MAPK and its upstream regulator ASK1 are involved in NO-induced PC12 cell death.

Animals↗
PubMed2005

Inhibition of MG132-induced mitochondrial dysfunction and cell death in PC12 cells by 3-morpholinosydnonimine.

The effect of 3-morpholinosydnonimine (SIN-1) against the cytotoxicity of MG132, a proteasome inhibitor, in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MG132 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS), and depletion of GSH. Addition of SIN-1, a producer of nitric oxide (NO) and superoxide, differentially reduced the MG132-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 150 microM. Carboxy-PTIO, superoxide dismutase, Mn-TBAP, and ascorbate prevented the inhibitory effect of SIN-1 on the cytotoxicity of MG132. SIN-1 inhibited the MG132-induced change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents in PC12 cells. S-nitroso-N-acetyl-DL-penicillamine reduced the MG132-induced cell death in PC12 cells, whereas peroxynitrite and H2O2 did not affect the cytotoxicity of MG132. The results suggest that NO and superoxide liberated from SIN-1 exert an inhibitory effect against the cytotoxicity of MG132. SIN-1 may inhibit the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability that is associated with oxidative damage.

Animals↗
PubMed2000

Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells.

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.

Adaptor Proteins, Signal Transducing↗
PubMed1996

BCR-ABL induces neurite-like structures and BCR lacking the SH2-binding domain induces cell rounding in PC12 cells.

The activated tyrosine kinase oncoprotein BCR-ABL is responsible for pathogenesis of Philadelphia chromosome-positive human leukemias. Because BCR carries a GAP (GTPase-activating protein) activity toward cytoskeleton-related small GTP-binding proteins, we utilized a neuronal PC12 cell system to test morphogenic potentials of BCR-ABL or BCR. We report here unique morphological phenotypes of PC12 cells expressing either BCR-ABL or a BCR mutant which lacks the SH2-binding domain (BCR Delta162-413). Although MAP kinase was not activated in PC12 cells expressing BCR-ABL, they showed incomplete neurite extensions even in the absence of the nerve growth factor (NGF). Overproduction of BCR Delta162-413 in PC12 cells, on the other hand, induced cell rounding in the absence of NGF. Interestingly, those cells could hardly make terminal differentiation in the presence of NGF and continued to grow without changing their round shape, although NGF receptor as well as MAP kinase appeared to be activated. Interestingly, the botulinum C3 toxin induced neurite-like structures in PC12 cells overexpressing BCR Delta162-413 without NGF.

Animals↗
PubMed2005

Involvement of p38 MAP kinase-mediated cytochrome c release on sphingosine-1-phosphate (S1P)- and N-monomethyl-S1P-induced cell death of PC12 cells.

d-erythro-Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, affects various neuronal functions including cell fate. S1P appears to have contradictory effects in PC12 cells, a neuronal model cell line; neurite retraction and cell survival/differentiation. In the present study, we examined whether S1P induces cell death in undifferentiated PC12 cells. Culture with S1P at 20 microM for 4 h caused lactate dehydrogenase leakage 24 h later. The response was reduced by an inhibitor of caspases and accompanied by the release of cytochrome c and DNA fragmentation. S1P caused the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) within 10 min. An inhibitor of p38 MAPK (10 microM SB203580) inhibited both the release of cytochrome c and DNA fragmentation induced by S1P. Treatment with nerve growth factor or pertussis toxin (PTX) decreased S1P-induced phosphorylation of p38 MAPK and cell death. These findings suggest that S1P-activated p38 MAPK acts as a death signal upstream of the release of cytochrome c. N-Monomethyl-S1P (MM-S1P), a weak agonist in cells expressing S1P1 receptors, had marked effects (phosphorylation of p38 MAPK, release of cytochrome c and DNA fragmentation) at lower concentrations than S1P and in a PTX-sensitive manner. These findings show that the activation of S1P receptors by S1P and MM-S1P causes cell death accompanied by DNA fragmentation via the p38 MAPK pathway-mediated release of cytochrome c in PC12 cells. The potential of S1P and MM-S1P to act as agonists of S1P receptors and as intracellular messengers is discussed.

Animals↗
PubMed2006

Cytoprotective effect of two synthetic enhancer substances, (-)-BPAP and (-)-deprenyl, on human brain capillary endothelial cells and rat PC12 cells.

Enhancer regulation is a new control mechanism in the brain [Knoll, J., 2003. Enhancer regulation/endogenous and synthetic enhancer compounds: a neurochemical concept of the innate and acquired drives. Neurochemical Research 28(8), 1275-1297]. Enhancer substances exert their effect in bi-modal form with a highly characteristic dose-dependency. Two bell-shaped concentration curves have been published. The one in ultra low concentration range (fM) specific form of enhancer regulation and the other at high concentration (100 microM) non-specific form of enhancer regulation. Catecholaminergic neurons proved to be enhancer-sensitive cells. Since rat PC12 cells and human brain endothelial cells (HBEC) work under catecholaminergic influence, it was reasonable to expect that both the specific and non-specific form of the enhancer regulation might be detectable in these cells. We tested this possibility on these cultured cells under normoxia and hypoxia-reoxygenation. After 1 h hypoxia produced by Argon gas and 0, 2, 4, and 20 h reoxygenation the cell loss was calculated by propidiumiodide assay and the cell activity was investigated by Alamar Blue assay colorimetric measurement. The percentages of living and necrotic cells were expressed after propidiumiodide staining. Broad scale concentrations of the two compounds (1 fM-100 microM) were added to the culture strait after the oxygen deprivation. (-)-BPAP and (-)-deprenyl, due to their enhancer effect, exerted a significant cytoprotective effect on both HBECs and PC12 cells. In harmony with Knoll's publications we were able to demonstrate by the aid of (-)-BPAP and (-)-deprenyl that both HBEC and PC12 are enhancer-sensitive cells. We detected the specific form of the enhancer regulation in the ultra low concentration range (fM-pM) and also the non-specific form of the enhancer regulation was visible (mM-microM).

Animals↗
PubMed2002

Differential effect of catecholamines and MPP(+) on membrane permeability in brain mitochondria and cell viability in PC12 cells.

The present study examined the effect of dopamine, 6-hydroxydopamine (6-OHDA), and MPP(+) on the membrane permeability transition in brain mitochondria and on viability in PC12 cells. Dopamine and 6-hydroxydopamine induced the swelling and membrane potential change in mitochondria, which was inhibited by addition of antioxidant enzymes, SOD and catalase. In contrast, antioxidant enzymes did not reduce the effect of MPP(+) on mitochondrial swelling and membrane potential. Catecholamines enhanced the Ca(2+) uptake and release by mitochondria, and the addition of MPP(+) induced Ca(2+) release. Catecholamines induced a thiol oxidation in mitochondria that was decreased by antioxidant enzymes. MPP(+) showed a little effect on the cytochrome c release from mitochondria and did not induce thiol oxidation. Catecholamines and MPP(+) induced a cell death, including apoptosis, in PC12 cells that was inhibited by addition of antioxidant enzymes. The result suggests that the oxidation of dopamine and 6-hydroxydopamine could modulate the membrane permeability in brain mitochondria and induce PC12 cell death, which may be ascribed to oxidative stress. MPP(+) appears to exert a toxic effect on neuronal cells by the action, which is different from catecholamines.

1-Methyl-4-phenylpyridinium↗