Wild-type PC12 Research Articles

Ca2+-dependent Activator Protein for Secretion 1 Is Critical for Constitutive and Regulated Exocytosis but Not for Loading of Transmitters into Dense Core Vesicles

Although CAPS1 was originally identified as a soluble factor that reconstitutes Ca(2+)-dependent secretion from permeabilized neuroendocrine cells, its exact function in intact mammalian cells remains controversial. Here we investigate the role for CAPS1 by generating stable cell lines in which CAPS1 is strongly down-regulated. In these cells, Ca(2+)-dependent secretion was strongly reduced not only of catecholamine but also of a transfected neuropeptide. These secretion defects were rescued by infusion of CAPS1-containing brain cytosol or by transfection-mediated expression of CAPS1. Whole cell patch clamp recording revealed significant reductions in slow burst and sustained release components of exocytosis in the knockdown cells. Unexpectedly, they also accumulated higher amounts of endogenous and exogenous transmitters, which were attributable to reductions in constitutive secretion. Electron microscopy did not reveal abnormalities in the number or docking of dense core vesicles. Our results indicate that CAPS1 plays critical roles not only in Ca(2+)-dependent, regulated exocytosis but also in constitutive exocytosis downstream of vesicle docking. However, they do not support the role for CAPS1 in loading transmitters into dense core vesicles.

Myc and E1A oncogenes alter the responses of PC12 cells to nerve growth factor and block differentiation.

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc or E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors and ornithine decarboxylase induction were similar in myc- and E1A-expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors and hormones.

P22PHOX KNOCKDOWN ATTENUATES NOREPINEPHRINE TRANSPORTER REDUCTION IN RESPONSE TO ENDOTHELIN‐1 IN PC12 CELLS

In DOCA-salt hypertension, superoxide anion (O2-·) levels are increased in sympathetic ganglion neurons as compared to normotensive controls; this increase is mediated in part by endothelin-1 (ET-1) via the activation of NADPH oxidase. Increased O2-· reduces norepinephrine uptake via norepinephrine transporter (NET). We tested the hypothesis that ET-1 reduces NET expression in PC12 cells via increases in O2-·, and this effect can be abolished by knockdown of p22phox, an indispensable component of the O2-·-generating NADPH oxidase. Undifferentiated PC12 cells were transfected with short hairpin RNA (shRNA) targeting p22phox. Cells with stable expression of p22phox shRNA (shRNA-p22) had reduced p22phox mRNA (71% knockdown) and protein (55% knockdown). Compared to wild type PC12 cells (WT), O2-· production induced by ET-1 was diminished in shRNA-p22. To assess ET-1 effects mediated by NADPH oxidase on NET, both shRNA-p22 and WT were exposed to ET-1 (100nM) for 30min to 24hrs. ET-1 reduced NET mRNA and protein levels in WT cells; the mRNA levels decreased over time and reached their minimum at 2 hrs (3.41 fold decrease, n=3), while protein levels remained reduced for 24 hrs (68% decrease, n=3). In shRNA-p22 cells, neither NET mRNA nor protein levels were changed by ET-1 treatment. These results indicate that the effects of ET-1 on O2-· production and NET expression in PC12 cells are p22phox -dependent.

L-NAME has Opposite Effects on the Productions of S-adenosylhomocysteine and S-adenosylmethionine in V12-H-Ras and M-CR3B-Ras Pheochromocytoma Cells

Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. Formed homocysteine is either catabolized into cystathionine (transsulfuration pathway) by cystathionine beta-synthase, or remethylated into methionine (remethylation pathway) by methionine synthase. To demonstrate the specificity of Ras-elicited effects on the activity of methyl cycles, wild-type pheochromocytoma PC12, mutant oncogenic rasH gene (MVR) expressing PC12 pheochromocytoma and normal c-rasH stably transfected M-CR3B cells were incubated with the N(omega)-nitro-L-arginine methyl ester (L-NAME), and manumycin, (inhibitors of nitric oxide synthase and farnesyltransferase, respectively). We have found that L-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was decreased. We conclude that depletion of endogenous NO with L-NAME increased the production of SAH only in cells with mutated oncogenic RasH, possibly through enhancement of production of reactive oxygen species (ROS). Oxidative stress can increase cystathionine beta-synthase activity that switches methyl cycles from remethylation into transsulfuration pathway to maintain the intracellular glutathione pool (essential for the redox-regulating capacity of cells) via an adaptive process.

Effects of RNA Interference of Atg4B on the Limited Proteolysis of LC3 in PC12 Cells and Expression of Atg4B in Various Rat Tissues

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC12 cells, for which the expression of Atg4B was 10% of wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-I. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, but the expression levels were not identical, but were dependent on the tissue, with the expression was high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.

Role of phospholipase C-gin NGF-stimulated differentiation and gene induction

The PC12 phaeochromocytoma cell line provides a useful model to study nerve growth factor-induced neuronal differentiation. The central signaling route of this process is mediated by the Ras-dependent extracellular signal-regulated kinase cascade. However, Ras-independent pathways are also stimulated by nerve growth factor and may contribute to differentiation signaling. One mediator for Ras-independent signal transduction in PC12 cells is phospholipase C-gamma that generates the second messengers diacylglycerol and inositol-trisphosphate. To probe the possible involvement of this enzyme in nerve growth factor-promoted differentiation, we used the phospholipase C inhibitor U73122 and the inositol-trisphosphate-receptor inhibitor Xestospongin C. Our results show that both chemicals block nerve growth factor-promoted neurite outgrowth, but the blockage of phospholipase C does not inhibit nerve growth factor-induced expression of c-fos, zif268 and transin genes. In addition, induction of these genes by nerve growth factor plus dibutyryl-cAMP is comparable in wild-type PC12 cells as well as in cells in which both Ras- and phospholipase C-gamma-mediated pathways are inhibited. The phospholipase C-gamma pathway thus belongs to those nerve growth factor receptor-originated signaling routes that contribute to the biological response of PC12 cells to nerve growth factor, but its gene activating potential does not have a major role in its neuritogenic effect.

α 2-Adrenergic receptors activate MAPK and Akt through a pathway involving arachidonic acid metabolism by cytochrome P450-dependent epoxygenase, matrix metalloproteinase activation and subtype-specific transactivation of EGFR

Previous study carried out on PC12 cells expressing each α 2-adrenergic receptor subtype individually (PC12/α 2A, /α 2B or /α 2C) have shown that epinephrine causes activation of PI3K and phosphorylation of Erk 1/2. The signal transduction mechanisms whereby each α 2-AR subtype triggers these actions were investigated in the present study. In all three clones, epinephrine-induced phosphorylation of MAPK or Akt was abolished by prior treatment with ketoconazole, but not with indomethacin or nordihydroguaiaretic acid. On the other hand, treatment of the clones with epinephrine caused a rapid increase of AA release, which was fully abolished by the PLC inhibitor U73122, but was unaffected by the PLA 2 inhibitor quinacrine. The effects of epinephrine on MAPK and Akt were mimicked by cell exposure to exogenous AA. Furthermore, whereas U73122 abolished the effects of epinephrine, quinacrine only prevented the effects of epinephrine, suggesting that AA release through PLC and its metabolites are responsible for MAPK and Akt activation by α 2-ARs. Treatment with 1,10-phenanthroline, CRM197, or tyrphostin AG1478 suppressed MAPK and Akt phosphorylation by epinephrine or AA, in a subtype-specific manner. Furthermore, conditioned culture medium from epinephrine-treated PC12/α 2 induced MAPK and Akt phosphorylation in wild-type PC12. Inhibition of NGFR tyrosine phosphorylation had no effect but the src inhibitor PP1 abolished MAPK and Akt phosphorylation in all three clones. Our results provide evidence for a putative pathway by which α 2-ARs activate MAPK and Akt in PC12 cells, involving stimulation of PLC, AA release, AA metabolism by cytochrome P450-dependent epoxygenase, stimulation of matrix metalloproteinases and subtype-specific transactivation of EGFR through src activation and heparin-binding EGF-like growth factor release.

Androgens activate mitogen‐activated protein kinase signaling: Role in neuroprotection

Recent evidence indicates that testosterone is neuroprotective, however, the underlying mechanism(s) remains to be elucidated. In this study, we investigated the hypothesis that androgens induce mitogen-activated protein kinase (MAPK) signaling in neurons, which subsequently drives neuroprotection. We observed that testosterone and its non-aromatizable metabolite dihydrotestosterone (DHT) rapidly and transiently activate MAPK in cultured hippocampal neurons, as evidenced by phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2. Importantly, pharmacological suppression of MAPK/ERK signaling blocked androgen-mediated neuroprotection against beta-amyloid toxicity. Androgen activation of MAPK/ERK and neuroprotection also was observed in PC12 cells stably transfected with androgen receptor (AR), but in neither wild-type nor empty vector-transfected PC12 cells. Downstream of ERK phosphorylation, we observed that DHT sequentially increases p90 kDa ribosomal S6 kinase (Rsk) phosphorylation and phosphorylation-dependent inactivation of Bcl-2-associated death protein (Bad). Prevention of androgen-induced phosphorylation of Rsk and Bad blocked androgen neuroprotection. These findings demonstrate AR-dependent androgen activation of MAPK/ERK signaling in neurons, and specifically identify a neuroprotective pathway involving downstream activation of Rsk and inactivation of Bad. Elucidation of androgen-mediated neural signaling cascades will provide important insights into the mechanisms of androgen action in brain, and may present a framework for therapeutic intervention of age-related neurodegenerative disorders.

Bcl-2, an Intrinsic Factor for Neuronal Regeneration

Two recent articles from the Chen lab investigate the intrinsic ability of neurons to regenerate and define the antiapoptotic protein Bcl-2 as a key player in this capability. Whether a neuron is capable of regenerating is only half of the story, though; the neuron must also be in a permissive environment. Understanding these two elements of neuronal regeneration can aid in developing therapies for the repair of central nerve damage due to injury or disease. Expression of bcl-2 is high in the embryonic central nervous system and decreases during development and acquisition of the nonregenerative phenotype. In the first article, Cho et al . show that if postnatal day 3 (P3) transgenic mice that overexpress Bcl-2 ( Bcl-2tg ) are subjected to optic nerve crush injury, the retinal ganglion cells (RGCs) regenerate and extend axons to the ipsilateral brain targets. (This is in contrast to the normal innervation path, which is to the contralateral side.) However, it was previously reported that if the injury occurs at P5, regeneration does not occur. Cho et al . show that RGCs from adult Bcl-2tg mice extended axons into embryonic brain slices, but this regenerative capacity diminished with the age of the target brain slice, such that essentially no neurites extended into the slices from animals older than P4. Thus, loss of regenerative capacity seems to reflect a target environment that is nonpermissive to growth, rather than an inability of the Bcl-2tg neurons to regenerate. Growth inhibition did not correlate with the appearance of myelin markers and was not prevented in mice deficient for CNS myelin; however, if 2- to 3-month-old wild-type or Bcl-2tg mice were treated with the astrocyte toxin L-a-aminoadipate immediately after optic nerve crush injury, then slight (wild-type) or robust (Bcl-2tg) nerve regeneration was observed. This and data from mice lacking glial fibrillary acidic protein (GFAP) and vimentin (two proteins associated with mature and embryonic glia, respectively) indicated that the glial response to injury in older brains is responsible for creating a nonpermissive environment for RGC regeneration. In the second article, Jiao et al . investigate the mechanism by which Bcl-2 allows neurons to retain their regenerative capacity. Optic nerves subjected to crush injury in P3 mice overexpressing Bcl-2 or the related protein Bcl-X L (Bcl-x L tg) showed decreased apoptosis compared with RGCs from wild-type mice; however, only the Bcl-2tg showed a regenerative response. Both Bcl-2-overexpressing and Bcl-x L tg-overexpressing cells exhibited enhanced survival compared with wild-type RGCs or PC12 cells, but only RGCs from Bcl-2tg mice or Bcl-2-transfected PC12 cells showed enhanced neurite extension in culture. Localization to the endoplasmic reticulum (ER) was necessary and sufficient for Bcl-2 or Bcl-X L (when targeted to the ER) to allow neurite outgrowth in response to growth factors in transfected PC12 cells. The ER is involved in calcium signaling, and PC12 cells overexpressing Bcl-2, which is normally targeted to the ER, showed decreased ER calcium storage based on cytosolic calcium concentration measurements made after depletion of the ER with the ER Ca 2+ -ATPase (SERCA) inhibitor thapsigargin. Indeed, a link between ER calcium concentration and neurite outgrowth was established by showing that overexpression of SERCA2b in PC12 cells reduced neurite outgrowth in the Bcl-2-overexpressing cells and that inhibition of SERCA increased neurite outgrowth. The authors propose that by inhibiting calcium reuptake into the ER by decreasing the abundance of SERCA, Bcl-2 increases the cytosolic calcium concentration during nerve injury. This was confirmed by measuring calcium concentration in the Bcl-2-overexpressing PC12 cells, which showed increased cytosolic calcium concentration in response to various stimuli. Furthermore, in the Bcl-2-overexpressing PC12 cells, there was enhanced phosphorylation of the transcriptional regulator CREB and the mitogen-activated protein kinase ERK and enhanced activation of CREB-reporter gene expression. Enhanced signaling in the ERK and CREB pathways was observed in the Bcl-2tg RGCs after optic nerve crush injury. Thus, Bcl-2 appears to have two roles in promoting axon regeneration: an antiapoptotic role and a neurite extension role related to the effect of Bcl-2 on calcium signaling in response to injury. K.-S. Cho, L. Yang, B. Lu, H. F. Ma, X. Huang, M. Pekny, D. F. Chen, Re-establishing the regenerative potential of central nervous system axons in postnatal mice. J. Cell Sci. 118 , 863-872 (2005). [Abstract] [Full Text] J. Jiao, X. Huang, R. A. Feit-Leithman, R. L. Neve, W. Snider, D. A. Dartt, D. F. Chen, Bcl-2 enhances Ca 2+ signaling to support the intrinsic regenerative capacity of CNS axons. EMBO J. 24 , 1068-1078 (2005). [Abstract] [Full Text]

Chronic ethanol exposure increases microtubule content in PC12 cells

BackgroundChronic ethanol exposure has been shown to result in changes in neuronal cyto-architecture such as aberrant sprouting and alteration of neurite outgrowth. In PC12 cells, chronic ethanol treatment produces an increase in Nerve Growth Factor (NGF)-induced neurite outgrowth that appears to require the epsilon, but not delta, isoform of Protein Kinase C (PKC). Neurites contain a core of microtubules that are formed from polymerization of free-tubulin. Therefore, it would be expected that an increase in neurite outgrowth would correlate with an increase in microtubule content. We examined the effect of chronic ethanol exposure on microtubule content in PC12 cells and the role of PKC epsilon and delta in ethanol's effect on microtubule levels.ResultsChronic ethanol exposure of wild-type and vector control PC12 cells resulted in a significant increase in microtubule content and a corresponding decrease in free tubulin. There was also a significant increase in microtubule content in PC12 cells expressing a dominate-negative inhibitor of epsilon PKC; cells which have previously been shown to have no ethanol-induced increase in neurite outgrowth. In contrast, ethanol had no effect on microtubule content in PC12 cells expressing a dominate-negative inhibitor of delta PKC.ConclusionThese results suggest that chronic ethanol exposure alters the relative ratio of free tubulin to microtubule-associated tubulin, an important component of the cytoskeleton. Further, the data from the PKC dominant-negative cell lines suggest that the effects of ethanol on microtubule content do not correlate with the effects of ethanol on neurite outgrowth. The delta isoform of PKC appears to be necessary for the ethanol-induced increase in microtubule content. These studies demonstrate an effect of chronic ethanol exposure which may contribute to previously documented alterations of neuronal cyto-architecture.

Synaptotagmin VII Is Targeted to Dense-core Vesicles and Regulates Their Ca2+-dependent Exocytosis in PC12 Cells

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.

Protein Kinase A Affects Trafficking of the Vesicular Monoamine Transporters in PC12 Cells

Previous studies have shown that the vesicular monoamine transporter 2 (VMAT2) is localized to both large dense core vesicles and synaptic vesicles in vivo. However, when exogenously expressed in PC12 cells, VMAT2 localizes only to large dense core vesicles. This distribution is similar to that of the endogenous vesicular monoamine transporter 1 (VMAT1) in PC12 cells. When VMAT2 was expressed in a protein kinase A (PKA)-deficient PC12 cell line it localized to synaptic-like microvesicles. Expression of recombinant VMAT1 in the same cell line showed a heterogeneous distribution to both large dense core vesicles and synaptic-like microvesicles. Coexpression of the PKA catalytic subunit partially restored trafficking of both VMAT2 and VMAT1 to large dense core vesicles; treatment of wild-type PC12 cells with the PKA inhibitor H89 increased VMAT2 on synaptic-like microvesicles. The VMAT1 and VMAT2 in large dense core vesicles exhibit a larger molecular size than those located on synaptic-like microvesicles. This difference is due to differential N-linked glycosylation. In vitro phosphorylation experiments show that PKA does not phosphorylate VMAT2. A chimera containing the VMAT2 cytoplasmic C-terminus fused to vesicular acetylcholine transporter (VAChT) shows mislocalization to synaptic-like microvesicles and VAChT-like glycosylation in the PKA-deficient cell line. However, coexpression with PKA changes the chimera's trafficking to large dense core vesicles and increases the molecular size. These results suggest that protein kinase A affects the formation and/or composition of VMAT trafficking complexes.

Nerve Growth Factor Withdrawal-mediated Apoptosis in Naïve and Differentiated PC12 Cells through p53/Caspase-3-dependent and -independent Pathways

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53, that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53, whereas other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53 has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal-mediated apoptosis in both naïve and differentiated PC12 cells. Our data show that as PC12 cells differentiate they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for naïve and 36 h for differentiated cells. Apoptosis involved caspases 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12[p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor withdrawal-mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.

Nuclear translocation of p90Rsk and phosphorylation of CREB is induced by ionomycin in a Ras-independent manner in PC12 cells.

In the present study we examined the possible role of p90Rsk in pathways leading to neuronal differentiation of PC12 cells induced by nerve growth factor (NGF) and the calcium ionophore ionomycin. PC12-M17 cells, expressing a dominant inhibitory Ras protein, do not undergo neuronal differentiation in response to NGF like wild-type PC12 cells, but exhibit neurite outgrowth when treated with NGF in combination with ionomycin. However, the blockade of Ras in these cells results in failure of activation of mitogen-activated protein kinase (MAPK)/extracellular signal regulation kinase (ERK) (MEK) and ERK activation as well, therefore kinases other than those of the ERK pathway might play a role in the induction of neuronal differentiation in this case. Here we show that p90Rsk translocates to the nucleus in response to ionomycin in both wild-type PC12 and PC12-M17 cells, and this spatial distribution is followed by increased phosphorylation of the cAMP response element binding protein (CREB). Since CREB is believed to be the transcription factor that can integrate Ca2+, growth factor and cAMP-induced signals, we suggest that p90Rsk may be one of the kinases which is able to replace ERKs under certain circumstances, thereby participating in Ras-independent neuronal differentiation induced by NGF plus ionomycin.

The fetal and neonatal brain protein neuronatin protects PC12 cells against certain types of toxic insult

The protein neuronatin is expressed in the nervous system of the fetus and neonate at a much higher level than in the adult. Its function is unknown. As a result of variable splicing, neuronatin mRNA exists in two forms, α and β. Wild type PC12 cells express neuronatin-α. We have isolated a PC12 variant, called 1.9, that retains many of the neuron-like properties of wild type PC12 cells, but it does not express neuronatin and it exhibits markedly increased sensitivity to the toxic effects of nigericin, rotenone and valinomycin. Pretreatment of the 1.9 cells with α-methyltyrosine, which inhibits dopamine synthesis, had little effect on the cells’ sensitivity to nigericin, rotenone or valinomycin indicating that dopamine-induced oxidative stress was not involved in the toxicity of these compounds. However, flattened cell subvariants of the 1.9 cells, which do not have any neuron-specific characteristics, did not exhibit increased sensitivity to nigericin indicating that some neuronal characteristic of the 1.9 cells contributed to the toxicity of nigericin. After the neuronatin-β gene was transfected into and expressed in the 1.9 cells, they regained wild type PC12 levels of resistance to nigericin, rotenone and valinomycin. These studies suggest that the function of neuronatin during development could be to protect developing cells from toxic insult occurring during that period.

Wnt‐1 dependent activation of the survival factor NF‐κB in PC12 cells

Expression of the Wnt-1 oncogene in PC12 cells induces morphological and biochemical changes, including up-regulation of cell adhesion and lack of differentiation in response to growth factors. The survival of PC12 cells is known to be mediated in part by phosphatidylinositol-3 kinase (PI-3 kinase)-dependent activation of the transcription factor nuclear factor-κB (NF-κB). We investigated the effect of Wnt-1 expression on cell survival and NF-κB activation using PC12 cells expressing Wnt-1 (PC12/Wnt1) and a reporter vector in which firefly luciferase expression is under the control of NF-κB consensus sequences. Serum deprivation caused apoptosis and decreased NF-κB activity in wild type PC12 cells. PC12/Wnt-1 cells showed less apoptosis in the absence of serum, and the levels of NF-κB activity were higher than in wild type PC12 cells. NF-κB activity was also increased by the transient expression of Wnt-1 in PC12 cells and it was completely inhibited in both PC12 and PC12/Wnt-1 cells by a dominant negative mutant IκB-α that has been shown to prevent NF-κB activation. Agents known to inhibit NF-κB-induced apoptosis in PC12 as well as in PC12/Wnt-1 cells, indicating a role of NF-κB activation in the anti-apoptotic effect of Wnt-1. Inhibition of PI-3 kinase with wortmannin, or with a dominant negative p85 regulatory subunit of the PI-3 kinase, blocked NF-κB activity in PC12 cells but caused only partial inhibition in PC12/Wnt-1 cells. The effect of Wnt-1 in activating NF-κB can be mimicked by inhibition of glycogen synthase kinase-3β (GSK-3β) with lithium or with a dominant negative GSK-3β. Our results show that expression of Wnt-1 increases survival of PC12 cells in the absence of serum by activating the anti-apoptotic factor NF-κB. Wnt-1-induced activation of NF-κB is partially independent of PI-3 kinase and can be mimicked by inhibition of GSK-3β. J. Neurosci. Res. 61:21–32, 2000. © 2000 Wiley-Liss, Inc.

Retroviral transfer of the ?-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor (NGF) was investigated. Differences in cell proliferation rate, neurite formation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for beta-galactosidase (beta-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuroblastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media obtained from NET cells infected with the NGF gene contained biologically active NGF, whereas media from beta-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate and increased neurite formation in wild-type, substrate-adherent PC-12 and Neuro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0-200 ng/ml) for 5 days significantly reduced proliferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with 125I-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET cells to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, beta-NGF gene transfer into wild-type NET cells induces the synthesis and secretion of NGF, temporarily decreases cell proliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation.

Retroviral transfer of the β-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor (NGF) was investigated. Differences in cell proliferation rate, neurite formation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for β-galactosidase (β-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuroblastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media obtained from NET cells infected with the NGF gene contained biologically active NGF, whereas media from β-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate and increased neurite formation in wild-type, substrate-adherent PC-12 and Neuro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0–200 ng/ml) for 5 days significantly reduced proliferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with 125I-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET cells to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, β-NGF gene transfer into wild-type NET cells induces the synthesis and secretion of NGF, temporarily decreases cell proliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation. J. Neurosci. Res. 59:265–275, 2000 © 2000 Wiley-Liss, Inc.

Wnt-1 dependent activation of the survival factor NF-kappaB in PC12 cells.

Expression of the Wnt-1 oncogene in PC12 cells induces morphological and biochemical changes, including up-regulation of cell adhesion and lack of differentiation in response to growth factors. The survival of PC12 cells is known to be mediated in part by phosphatidylinositol-3 kinase (PI-3 kinase)-dependent activation of the transcription factor nuclear factor-kappaB (NF-kappaB). We investigated the effect of Wnt-1 expression on cell survival and NF-kappaB activation using PC12 cells expressing Wnt-1 (PC12/Wnt1) and a reporter vector in which firefly luciferase expression is under the control of NF-kappaB consensus sequences. Serum deprivation caused apoptosis and decreased NF-kappaB activity in wild type PC12 cells. PC12/Wnt-1 cells showed less apoptosis in the absence of serum, and the levels of NF-kappaB activity were higher than in wild type PC12 cells. NF-kappaB activity was also increased by the transient expression of Wnt-1 in PC12 cells and it was completely inhibited in both PC12 and PC12/Wnt-1 cells by a dominant negative mutant IkappaB-alpha that has been shown to prevent NF-kappaB activation. Agents known to inhibit NF-kappaB-induced apoptosis in PC12 as well as in PC12/Wnt-1 cells, indicating a role of NF-kappaB activation in the anti-apoptotic effect of Wnt-1. Inhibition of PI-3 kinase with wortmannin, or with a dominant negative p85 regulatory subunit of the PI-3 kinase, blocked NF-kappaB activity in PC12 cells but caused only partial inhibition in PC12/Wnt-1 cells. The effect of Wnt-1 in activating NF-kappaB can be mimicked by inhibition of glycogen synthase kinase-3beta (GSK-3beta) with lithium or with a dominant negative GSK-3beta. Our results show that expression of Wnt-1 increases survival of PC12 cells in the absence of serum by activating the anti-apoptotic factor NF-kappaB. Wnt-1-induced activation of NF-kappaB is partially independent of PI-3 kinase and can be mimicked by inhibition of GSK-3beta.

Activation of c-Ha-Ras by Nitric Oxide Modulates Survival Responsiveness in Neuronal PC12 Cells

p21(c-Ha-Ras) (Ras) can be activated by the guanine nucleotide exchange factor mSOS1 or by S-nitrosylation of cysteine 118 via nitric oxide (NO). To determine whether these two Ras-activating mechanisms modulate distinct biological effects, a NO-nonresponsive Ras mutant (Ras(C118S)) was stably expressed in the PC12 cells, a cell line that generates NO upon nerve growth factor treatment. We report here that Ras(C118S) functions indistinguishably from wild type Ras in activating and maintaining the mSOS1- and Raf-1-dependent mitogen-activated protein kinase cascade necessary for neuronal differentiation. However, continuous (>5 days) exposure to nerve growth factor reveals that, in contrast to parental or wild-type Ras-overexpressing PC12 cells, Ras(C118S)-expressing PC12 cells cannot sustain the basal interaction between Ras and phosphatidylinositol 3-kinase. This results in spontaneous apoptosis of these cells despite the presence of nerve growth factor and serum. Thus unique downstream effector interactions and biological outcomes can be differentially modulated by distinct modes of Ras activation.