ProNGF inhibits NGF-mediated TrkA activation in PC12 cells

Degeneration of cholinergic basal forebrain neurons (CBFN) is a hallmark in the pathology of Alzheimer's disease (AD). Critically depending upon the neurotrophic support through nerve growth factor (NGF), CBFN in the AD brain face elevated concentrations of the pro-form of NGF (proNGF) and suffer from an imbalance between TrkA and p75(NTR) expression. Research for the underlying mechanisms of CBFN death suggested a pro-apoptotic activity of proNGF. However, this finding could not be confirmed by all investigators and other studies even observed a neurotrophic function of proNGF. In the presence of these controversial findings we investigated the activity of proNGF in PC12 cells with specific emphasis on its neurotoxic versus neurotrophic action. In this study, we show that proNGF can mediate TrkA receptor signaling directly, yet in the manner of a partial agonist with a lower maximum activity than NGF. A pro-apoptotic activity of proNGF could not be confirmed in our cellular system. Interestingly and surprisingly, pre-incubation with proNGF at low, sub-active concentrations inhibited TrkA-mediated neurotrophic NGF signaling in PC12 cells. Our data support a novel hypothesis for the role of elevated proNGF levels in CBFN pathology in AD. Thus, proNGF can indirectly contribute to the slow neurodegeneration in AD by reducing NGF-mediated trophic support.     

Cyclooxygenase-2 expression inhibits trophic withdrawal apoptosis in nerve growth factor-differentiated PC12 cells

Cyclooxygenase-2 (Cox-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being up-regulated by diverse stimuli. Significantly Cox-2 mRNA is absent from rat pheochromocytoma (PC12) cells, both basally and following stimulation with a range of agonists. Using PC12 cells engineered to stably express isopropyl-1-thio-beta-D-galactopyranoside-inducible Cox-2 (PCXII-4), we have investigated the putative effects of Cox-2 expression on differentiation, proliferation, and trophic withdrawal apoptosis. Cox-2 bioactivity had no effect on nerve growth factor-induced differentiation, epidermal growth factor-induced proliferation, or aromatic L-amino acid decarboxylase expression. However, trophic withdrawal apoptosis, induced by the removal of nerve growth factor following differentiation, was markedly reduced in the PCXII-4 when compared with control cells, as assessed by annexin V staining, DNA laddering, and Hoechst 33258 staining. The specificity of this effect was confirmed using two pharmacologically distinct nonsteroidal anti-inflammatory drugs, indomethacin and NS398. Investigations showed that the activity of the pro-apoptotic protease caspase-3 was reduced in PCXII cells. This study demonstrates that Cox-2-derived prostaglandins exert cytoprotective effects in trophic factor withdrawal apoptosis and provides evidence that this is, at least in part, due to suppression of caspase-3 activity.     

Iron enhances NGF-induced neurite outgrowth in PC12 cells

Rat pheochromocytoma 12 (PC12) cells undergo neuronal differentiation in response to nerve growth factor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular signal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting effect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF-induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.     

新霉素抑制PC12细胞烟碱受体介导的反应

新霉素是一种氨基甙类抗生素,在细胞水平可以抑制磷脂酶C介质的信号转导系统,本研究采用全细胞膜片钳技术,以大鼠肾上腺嗜铬细胞瘤细胞（PC12)为标本,观察了新霉素参考书国酰胆碱诱发电流（IACh)的影响,药理学鉴定表明,PC12细胞上的IACh是通过ACh激动烟碱受体引起的,钳制电压为－80mV时,ACh(30umol/L)诱发一内向电流;细胞外同时给予新霉素（0．01－1mmol/L)和ACh(30μmol/L)可显著抑制IACh峰值,此抑制作用迅速,可逆,呈浓度依赖性,用新霉素预处理细胞3－8min不影响其对IACh的抑制作用,用外源性蛋白激酶C（PKC）激剂激活PKC,同样可抑制IACh,而细胞内透析PKC抑制剂（PKCI19-31,0.1-5μmol/L）不影响新霉素对IACh的抑制作用,以上结果提示,新霉对PC12细胞的IACh的有抑制作用,这是一种与磷脂酶C阻断无关的药理学效应。     

Adenosine inhibits cell division and promotes neurite extension in PC12 cells

Low concentrations (10-50 microM) of adenosine (EC50 = 17 microM) or chloroadenosine (EC50 = 23 microM) prevent the division of PC12 cells. This inhibition is not mimicked by guanosine, inosine, 3',5' dideoxyadenosine, phenylisopropyladenosine, or adenylylimidodiphosphate. The growth inhibition is not relieved by addition of uridine or deoxycytidine, nor is it potentiated by homocysteine thiolactone. Inhibition of adenosine uptake does not inhibit adenosine-dependent growth arrest. PC12 variants that are deficient in adenosine kinase are as sensitive as wild-type cells to the growth-inhibitory effects of adenosine. These experiments suggest that adenosine prevents cell division at an adenosine receptor rather than acting after being metabolically altered. The adenosine receptor that inhibits cell division does not appear to be the adenosine receptor that stimulates adenylate cyclase for these reasons: (1) phenylisopropyladenosine, which is a potent agonist of this receptor, does not inhibit cell division; (2) 3',5' dideoxyadenosine does not antagonize the effect of adenosine on cell division; and (3) theophylline does not affect growth inhibition by adenosine. Thus, these experiments suggest the existence of a second adenosine receptor that can inhibit cell division. Adenosine also promotes the morphological differentiation of PC12 cells. In the presence of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenosine (EHNA), adenosine causes the formation of short neurites (one-half to one and one-half cell diameters in length). Adenosine also increases the rate of neurite formation of both long and short neurites in response to NGF.     

Clustering-induced signaling of CEACAM1 in PC12 cells

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an Ig-like transmembrane protein, functions in cell adhesion, angiogenesis and epithelial cell morphogenesis, and has been identified as a tumor suppressor. For all of these functions, CEACAM1 requires signaling capabilities. However, the mechanisms of CEACAM1-mediated signaling are only poorly understood. Here we characterized for the first time CEACAM1 expression and signaling in the neuroendocrine rat pheochromocytoma PC12 cell line. Stimulation of CEACAM1 by ligation on the cell surface with antibodies induced formation of large CEACAM1 clusters and a rapid and transient CEACAM1 tyrosine dephosphorylation. Functionally, this dephosphorylation correlated with a reduced association between CEACAM1 and the tyrosine phosphatase SHP2. Clustering also stimulated binding of CEACAM1 to the actin cytoskeleton, measured by a partial translocation of CEACAM1 into the insoluble fraction after detergent extraction. Both tyrosine dephosphorylation and interaction with the cytoskeleton were sensitive to neuronal differentiation of PC12 cells. The first detected downstream activation of the mitogen-activated protein kinases ERK1 and ERK2, but not of JNK or p38, describes a novel target of CEACAM1-mediated signaling and contributes to the understanding of how CEACAM1 regulates cellular function.     

Cyclic AMP-elevating agents induce the expression of MAP kinase phosphatase-1 in PC12 cells

Stimulation of pheochromocytoma PC12 cells by cAMP-elevating agents caused the induction of the immediate early gene 3CH134, which encodes MAP kinase phosphatase-1 (MKP-1). Forskolin was as potent as serum in stimulating MKP-1 gene expression, whereas dibutyryl-cAMP and neuropeptide PACAP were less effective. Induction of the MKP-1 gene was accompanied by neo-synthesis of MKP-1 protein. MAP kinase activation was not involved in the cAMP-induced MKP-1 gene expression. The MAP kinase inactivation, that would result from MKP-1 induction in response to increased intracellular cAMP level, contributes to explain how hormones or neurotransmitters signaling through cAMP influence cell growth and differentiation.     

Efficient transfection and expression of heterologous genes in PC12 cells

The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.     

Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells

Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of Parkinson's disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis.     

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

Synaptotagmin VII (Syt VII), which has a higher Ca²⁺ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca²⁺-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca²⁺ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.     

Overexpression of complexin in PC12 cells inhibits exocytosis by preventing SNARE complex recycling

Complexin is an important protein that functions during Ca2+-dependent neurotransmitter release. Substantial evidence supports that complexin performs its role through rapid interaction with SNARE complex with high affinity. However, alpha-SNAP/NSF, which can disassemble the cis-SNARE complex in the presence of MgATP, competes with complexin to bind to SNARE complex. In addition, injection of alpha-SNAP into chromaffin cells enhances the size of the readily releasable pool, and mutation disrupting the ATPase activity of NSF results in the accumulation of SNARE complex. Thus, whether high concentrations of complexin could result in a reverse result is unclear. In this paper, we demonstrate that when stably overexpressed in PC12 cells, high levels of complexin result in the accumulation of SNARE complex. This in turn leads to a reduction in the size of the readily releasable pool of large dense core vesicles. These results suggest that high levels of complexin seem to prevent SNARE complex recycling, presumably by displacing NSF and alpha-SNAP from SNARE complex.     

Regulatory mechanism of PAC1 gene expression via Sp1 by nerve growth factor in PC12 cells

In addition to VPAC1 and VPAC2, PAC1 is involved in the pleiotropic action of pituitary adenylate cyclase activating polypeptide (PACAP) in the CNS. A luciferase reporter assay for the human PAC1 gene (-2160/+268) revealed that NGF treatment significantly augments the promoter activity of the PAC1 gene. Moreover, the Sp1 site at -282/-273 was shown to be essential for the NGF-augmented promoter activity of the PAC1 gene. Treatment with U0126, an MEK inhibitor, or Mithramycin A, an Sp1 inhibitor, significantly attenuated promoter activity. These results indicate that activation of Sp1 by the Ras/MAPK pathway might participate in neuron specific expression of the PAC1 gene.     

Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells

Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.