Combinatorial code of growth factors and neuropeptides define neuroendocrine differentiation in PC12 cells

Adrenal chromaffin cells constitute one of the first cell types to have been defined as a neuroendocrine cell type. Since they produce dopamine, these cells have been proposed for the treatment of neuronal deficits in human Parkinson's disease. However, the factors involved in the development of chromaffin cells are still poorly understood. Based on recent insights from stem cell research, we decided to study the role of extracellular matrices, growth factors and neuropeptides on the neuroendocrine differentiation in a serum-free medium of PC12 cells. Employing immunohistochemistry, quantitative PCR and HPLC analysis, neuroendocrine differentiation was determined by evaluating neurite outgrowth, catecholamine biosynthesis and release as well as neuropeptide and vesicular protein mRNA expression. The combination of bFGF, NGF and PACAP could prevent the inhibition of neurite process development induced by dexamethasone in PC12 cells cultured on ECM. Whereas glucocorticoids were essential in the regulation of enzymes of catecholamine biosynthesis and metabolism, growth factors and PACAP were more efficient in inducing neuropeptide and chromogranin B expression as well as release of dopamine and 3-methoxytyramine. Therefore, in addition to glucocorticoids, chromaffin cells need a gradient of matrix, growth factors, and neuropeptides to develop the full functional phenotype of a neuroendocrine cell.     

Subcellular localization of epitope-tagged neurotrophins in neuroendocrine cells

A growing body of evidence suggests that neurotrophins (NTs) play a critical role in synaptic plasticity and other activity dependent processes in the CNS. Release of these growth factors by neurons and neuroendocrine cells was recently shown to occur via the regulated secretory pathway, representing a possible mechanism for preferentially supplying NTs locally to active synapses. However, the identity and characteristics of the intracellular storage compartment for NTs undergoing stimulus-coupled secretion remains controversial. As a step towards addressing these issues we have investigated the subcellular localization of epitope-tagged nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in neuroendocrine cells. Placement of the myc-epitope tag at the neurotrophin carboxy terminus did not affect essential properties of the NTs such as their ability to induce Trk tyrosine phosphorylation or their sorting into the regulated secretory pathway in PC12 and AtT-20 neuroendocrine cells. Epitope-tagged NTs colocalize with dense core vesicle (DCV)-markers at the light microscopic level in both cell lines investigated. Furthermore, at an EM level immunoreactivity (IR) for myc-tagged NGF was found over dense core granules (DCGs) in PC12 cells. These data provide evidence that NTs can be stored in DCVs in neuronal model cell lines and, potentially, in neurons as well. J. Neurosci. Res. 51:463–472, 1998. © 1998 Wiley-Liss, Inc.     

Implication of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) for Neuroprotection of Nicotinic Acetylcholine Receptor Signaling in PC12 Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neurotrophic factor through PAC1-R, PACAP-specific receptor, in the central nervous system. On the other hand, by interacting with nicotinic acetylcholine receptor (nAChR), nicotine exhibits several neuroprotective effects. Since the relevance of PACAP and nAChR signaling has not been reported so far, we attempted to investigate their relevance in terms of neuroprotection in PC12 cells. Regarding the effect of nicotine on PACAP gene expression, nicotine increased its mRNA level in time-dependent and dose-dependent manners in the PC12 cells differentiated with nerve growth factor (NGF). In addition, luciferase reporter assay demonstrated that nicotine treatment significantly augments the promoter activity of PACAP gene. Since PAC1-R mRNA expression was induced by NGF, PACAP exhibited neuroprotective effect against tunicamycin-induced cell death in the differentiated PC12 cells. Nicotine also exhibited the neuroprotective effect, which was significantly attenuated by Max.d.4 (PAC1-R specific antagonist). These results suggest that the increment of PACAP gene expression due to nicotine treatment might be involved in the neuroprotection by nicotine.     

Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. I. Stimulation of hydrogen peroxide resistance

The nerve growth factor protein (NGF) regulates neuronal cell death during the development of embryonic sensory and sympathetic neurons in the peripheral nervous system (PNS). NGF protects the rat pheochromocytoma line PC12, a useful model of NGF responsive peripheral neurons, from hydrogen peroxide, which interacts with ferrous iron to generate hydroxyl radicals. Exogenous catalase provides protection, whereas superoxide dismutase (SOD) has no effect on neuronal survival when PC12 cells are challenged with hydrogen peroxide. NGF treatment of PC12 cells increases the activity of catalase. NGF protection from hydrogen peroxide is partially abolished by aminotriazole (Az), a low molecular weight catalase inhibitor. Taken together, these data are consistent with the hypothesis that NGF protects from peroxidative events and consequent cell death via an induction of free radical detoxifying mechanisms, such as catalase activity.     

Dimethylarginine dimethylaminohydrolase 1 regulates nerve growth factor-promoted differentiation of PC12 cells in a nitric oxide-dependent but asymmetric dimethylargenine-independent manner

There are significant morphological and biochemical alterations during nerve growth factor (NGF)-promoted neuronal differentiation, and the process is regulated by molecules, including nitric oxide (NO). Dimethylarginine dimethylaminohydrolase (DDAH) is thought to play a critical role in regulating NO production via hydrolyzing the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Thus, we tested the role of DDAH in NGF-promoted differentiation of PC12 (pheochromocytoma) cells. The present results show that both mRNA and protein levels of DDAH1 were increased, whereas those of DDAH2 were decreased, during NGF-promoted cell differentiation. Both the DDAH activity and the ADMA level in cultured medium were unchanged in this process. NGF promoted neurite formation and induced the expression of microtubule-associated protein 2 (MAP2), a neuronal marker, which were both significantly repressed by DDAH1 silence with small interfering RNA but not by DDAH2 silence. The expressions of three isoforms of NOS were markedly upregulated after NGF stimulation with a time course similar to that of DDAH1, which were attenuated by DDAH1 silence. Conversely, overexpression of DDAH1 accelerated neurite formation in PC12 cells, concomitantly with upregulating the expression of three NOS isoforms. In summary, our data reveal the critical regulatory effect of DDAH1 on NGF-promoted differentiation of PC12 cells in an NOS/NO-dependent but ADMA-independent manner.     

PC3 potentiates NGF-induced differentiation and protects neurons from apoptosis

PC3TIS21/BTG2 is member of a novel family of antiproliferative genes (BTG1, ANA/BTG3, PC3B, TOB, and TOB2) that play a role in cellular differentiation. We have previously shown that PC3TIS21/BTG2 is induced by nerve growth factor (NGF) at the onset of neuronal differentiation in the neural crest-derived PC12 cell line, and is a marker for neuronal birth. We now observe that PC3TIS21/BTG2 ectopically expressed in PC12 cells synergises with NGF, similarly to the cyclin-dependent kinase inhibitor p21, potentiating the induction of the neuronal markers tyrosine hydroxylase and neurofilament 160 kDa. Furthermore, PC3TIS21/BTG2 protects from apoptosis elicited by NGF deprivation in terminally differentiated PC12 cultures. Such effects might be a consequence of the arrest of cell cycle exerted by PC3TIS21/BTG2, or expression of a sensitizing (neurogenic) property of the molecule.     

NOGO—A在PC12细胞分化过程中的表达及意义

目的探讨NOGO—A在PC12细胞生长发育过程中的表达及意义。方法培养大鼠嗜铬细胞瘤细胞系PC12细胞，用神经生长因子诱导其分化，并于倒置显微镜下随机取20视野计数观察细胞增值和轴突生长情况。采用免疫荧光染色、逆转录酶聚合酶链反应（RT—PCR）及免疫印迹法等方法检测诱导后第1d、第3d、第5d、第7d PC12细胞中NOGO—AmRNA及蛋白的表达及变化，并留取细胞培养液检测多巴胺水平。结果未分化的PC12细胞中未检测到NOGO—A mRNA及蛋白表达。经神经生长因子诱导的PC12细胞，细胞轴突不断生长，NOGO—AmRNA及蛋白的表达逐渐增高（P〈0．05）。PC12细胞在分化过程中多巴胺（DA）分泌水平无明显差别。结论PC12细胞向交感神经元分化的过程中NOGO—A的表达逐渐增强，推测NOGO—A在神经元发育早期可能促进轴突生长。但对多巴胺激素释放的调节不明显。     

Purkinje cell protein 4 positively regulates neurite outgrowth and neurotransmitter release

Purkinje cell protein 4 (PCP4), also called brain-specific polypeptide 19 (PEP19), is a neurospecific, small calmodulin-binding protein that binds both calcium-free and calcium-binding calmodulin to regulate the calmodulin-mediated signal. The expression level of this molecule is decreased in the brain in Alzheimer's disease, Huntington's disease, and alcoholism. However, little is known of the function of PCP4 regarding neuronal or neuroendocrine cell differentiation and neurotransmitter release. To address this, we established a PCP4 tetracycline-inducible rat chromaffin cell line, PC12. When PCP4 expression was induced with doxcycline, neurite outgrowth was significantly advanced in the presence of nerve growth factor (NGF) and dibutyryl cAMP, which was inhibited by W-7, a calmodulin inhibitor, and PD98059, an ERK inhibitor. In addition, size of the cell body also was increased by treatment with NGF in the PCP4-induced PC12 cells. Constitutive and potassium-evoked release of acetylcholine and dopamine was increased and apoptosis induced by hydrogen peroxide (H(2)O(2)) was inhibited in PCP4-induced PC12 cells. On the other hand, knockdown of PCP4 by siRNA transfection decreased neurite outgrowth and dopamine release and increased H(2)O(2)-induced apoptosis in PC12 cells. These results indicate that PCP4 promotes neuroendocrine cell differentiation and neurotransmitter release by activating calmodulin function.