Multiple signaling conduits regulate global differentiation-specific gene expression in PC12 cells

PC12 cells serve as a model for exploring nerve growth factor (NGF)-stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF-stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin-like growth factor-1 (IGF-1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF-1. From the set of NGF-specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra-1 and transforming growth factor beta 1 (TGF beta 1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation-dependent protein (RGT), and synapsin II required neither mitogen-activated protein kinase (MAPK) pathway. NGF-induction of the bradykinin B2 receptor and c-Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK-dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK-independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF-dependent gene expression, but additional Ras-dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation-specific gene expression in PC12 cells.     

Stimulation of expression for the adenosine A2A receptor gene by hypoxia in PC12 cells. A potential role in cell protection.

The purpose of this study was to examine the regulation of adenosine A2A receptor (A2AR) gene expression during hypoxia in pheochromocytoma (PC12) cells. Northern blot analysis revealed that the A2AR mRNA level was substantially increased after a 3-h exposure to hypoxia (5% O2), which reached a peak at 12 h. Immunoblot analysis showed that the A2AR protein level was also increased during hypoxia. Inhibition of de novo protein synthesis blocked A2AR induction by hypoxia. In addition, removal of extracellular free Ca2+, chelation of intracellular free Ca2+, and pretreatment with protein kinase C inhibitors prevented A2AR induction by hypoxia. Moreover, depletion of protein kinase C activity by prolonged treatment with phorbol 12-myristate 13-acetate significantly inhibited the hypoxic induction of A2AR. A2AR antagonists led to a significant enhancement of A2AR mRNA levels during hypoxia, whereas A2AR agonists caused down-regulation of A2AR expression during hypoxia. This suggests that A2AR regulates its own expression during hypoxia by feedback mechanisms. We further found that activation of A2AR enhances cell viability during hypoxia and also inhibits vascular endothelial growth factor expression in PC12 cells. Thus, increased expression of A2AR during hypoxia might protect cells against hypoxia and may act to inhibit hypoxia-induced angiogenic activity mediated by vascular endothelial growth factor.     

Comparison of the expression of cell surface poly-N-acetyllactosamine-type oligosaccharides in PC12 cells with those in its variant PC12D.

To explore the biological role of carbohydrate chains in the process of nerve cell differentiation, we carried out a characterization of the carbohydrate structure of glycoproteins by comparing conventional PC12 cells with variant cells (PC12D). In vitro metabolic labeling of cells with either [(3)H] glucosamine or [(3)H] threonine, together with tomato lectin staining, revealed that nerve growth factor (NGF) stimulation caused a decrease in the poly-N-acetyllactosamine synthesis of high-molecular-weight glycopeptides from PC12 cells. By comparison, the amount of glycopeptides with poly-N-acetyllactosamine from PC12D cells was already significantly low and it was not changed by NGF stimulation. By assaying the glycosyltransferases that participate in poly-N-acetyllactosamine synthesis, the decrease in the amount of the poly-N-acetyllactosamine in PC12D cells as well as NGF-stimulated PC12 cells could be accounted for by a reduction in the activity of poly-N-acetyllactosamine extension enzyme (GnT-i), because the amount of poly-N-acetyllactosamine in both cells precisely correlated with changes in GnT-i activity, whereas the activities of N-acetylglucosaminyltransferase V (GnT-V) and beta 1-4 galactosyltransferase remained unchanged. These results demonstrate that the decrease in poly-N-acetyllactosamine synthesis in PC12 cells occurred prior to neurite formation, whereas PC12D cells were insensitive to this effect. Next, we showed that GnT-i but not GnT-V catalyzed a rate-limiting reaction in the expression of poly-N-acetyllactosamine chains, especially in pheochromocytoma.     

Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC12 cells

We studied the regulation of glucose-6-phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose- and time-dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia-induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia-inducible factor (HIF)-dependent glycolytic enzyme. Hypoxic-G6PD induction was almost negligible in non-excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO(2). Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N-acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose-metabolizing enzymes.     

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.     

Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis

Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled. Proteomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 μM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and calcium-binding protein 1 were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.     

N-CAM expression and localization in PC12 cells modulated by extracellular peptides

The neural cell adhesion molecules (N-CAMs) play an important role in mediating cell–cell interactions in the nervous system. Different isoforms of these membrane proteins are involved in the formation of the neuronal network and in the dynamic phases of neuronal plasticity.

We studied the early stages of the pseudo neuronal differentiation of PC12 cells induced by a class of small acidic peptides capable of modulating gene expression in these cells.

The data presented here indicate that peptides with specific sequences induce an increase in N-CAM mRNA expression and protein translocation to the plasma membrane to a comparable degree as NGF.     

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.     

Regulation of gene expression in PC12 cells via an activator of dual second messengers: pituitary adenylate cyclase activating polypeptide.

In this study we demonstrate that the activator protein-1 (AP-1) DNA motif, initially considered to be unresponsive to cyclic AMP (cAMP), does function as a cAMP-response element in PC12 cells. A luciferase reporter gene driven by the collagenase promoter that contains the AP-1 motif is responsive to cAMP as well as phorbol esters when transfected in PC12 cells. We have recently shown that pituitary adenylate cyclase activating peptide (PACAP) has neurotrophic properties and activates both adenylylcyclase and the inositol lipid cascade in PC12 cells. Consistent with these actions, we demonstrate that PACAP is an effective activator of luciferase reporter genes whose promoters bear the AP-1 motif, as well as the related DNA element that binds the protein CREB. Both the cAMP and inositol lipid pathways appear to play a role in the activation of these motifs by PACAP. Mutation of the AP-1 motif and its juxtaposition to a heterologous promoter proves that the AP-1 motif is a locus for response to cAMP and PACAP. The luciferase reporter genes bearing the AP-1 motif are not cAMP responsive in HeLa tk- cells, indicating that the mode of second-messenger responsiveness is cell-type specific.