N-acetylglucosaminyltranferase VB expression enhances beta1 integrin- dependent PC12 neurite outgrowth on laminin and collagen

N-acetylglucosaminyltransferase VB (GnT-VB, -IX) is a newly discovered glycosyltransferase expressed exclusively in high levels in neuronal tissue during early development. Its homolog, GnT-V, is expressed in many tissues and modulates cell-cell and cell-matrix adhesion. The ability of GnT-VB to regulate cell-matrix interactions was initially investigated using the rat pheochromocytoma PC12 neurite outgrowth model. PC12 cells stably transfected with GnT-VB consistently showed an enhanced rate of nerve growth factor (NGF)-induced neurite outgrowth on collagen and laminin substrates. Levels of TrkA receptor phosphorylation and downstream ERK activation induced by NGF were not influenced by GnT-VB expression. No significant difference was observed in the rate of neurite outgrowth when cells were cultured on non-coated culture dishes, indicating that integrin-ECM interaction is required for the stimulatory effects. Neurite outgrowth induced by manganese-dependent activation of beta1 integrin on collagen and laminin substrates, however, showed a significant increase in neurite length for the PC12/GnT-VB cells, compared with control cells, suggesting that the enhancement is most likely mediated by alteration of beta1 integrin-ECM interaction by GnT-VB. These results demonstrate that GnT-VB expression can modulate the rate of neurite outgrowth by affecting beta1 integrin-ECM interaction.     

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras,or src tyrosine kinase

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.     

The changes in the neuronal PC12 and the intestinal epithelial Caco-2 cells during the coculture. The functional analysis using an in vitro coculture system

The interaction between intestinal epithelial cells andperipheral neuronal cells were examined using an invitro coculture system. Two cell lines, Caco-2 and PC12, were usedfor this experiment as an intestinal epithelial and entericneuronal cell model, respectively. By coculturing with fullydifferentiated Caco-2 cells, the neurite outgrowth was inducedin PC12 cells. This neurite outgrowth in PC12 was blocked byanti-nerve growth factor (NGF) polyclonal antibodies,suggesting that the neurite outgrowth in PC12 during thecoculture with Caco-2 cells was due to NGF secreted fromCaco-2 cells. On the other hand, coculturing with fullydifferentiated PC12 cells induced the decrease oftransepithelial electrical resistance in Caco-2 cellmonolayers. The permeability of lucifer yellow alsosignificantly increased, suggesting that the barrier functionand paracellular permeability of Caco-2 monolayers werealtered by coculturing with PC12 cells. The present studysuggests that this in vitro coculture system is a good modelfor the functional analysis of interaction among intestinalepithelial cells with different cell types.     

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

Reconstituted basement membrane enhances neurite outgrowth in PC12 cells induced by nerve growth factor

Rat pheochromocytoma cells (PC12) respond to nerve growth factor (NGF) by elaborating neurites. We have studied the effect of extracellular matrix proteins on responsiveness of PC12 cells to NGF. NGF elicited neurite outgrowth in cells plated on collagen, fibronectin, or laminin within 48-72 h. In contrast, cells entrapped in reconstituted basement membrane prepared from the Engelbreth-Holm-Swarm tumor (EHS-BM) responded within 24 h. Cells plated on collagen or fibronectin required a minimal dose of 50 ng/ml NGF to achieve the same effect as cells entrapped in EHS-BM at 1 ng/ml NGF. Neurites displayed by cells plated in EHS-BM were more extensively branched and remained intact up to 21 days following a single administration of NGF.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

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.     

Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells

Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.     

The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Cell aggregation and neurite growth in gels of extracellular matrix molecules

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.     

The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner

The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.     

SH2B1beta (SH2-Bbeta) enhances expression of a subset of nerve growth factor-regulated genes important for neuronal differentiation including genes encoding urokinase plasminogen activator receptor and matrix metalloproteinase 3/10

Previous work showed that the adapter protein SH2B adapter protein 1beta (SH2B1) (SH2-B) binds to the activated form of the nerve growth factor (NGF) receptor TrkA and is critical for both NGF-dependent neurite outgrowth and maintenance. To identify SH2B1beta-regulated genes critical for neurite outgrowth, we performed microarray analysis of control PC12 cells and PC12 cells stably overexpressing SH2B1beta (PC12-SH2B1beta) or the dominant-negative SH2B1beta(R555E) [PC12-SH2B1beta(R555E)]. NGF-induced microarray expression of Plaur and Mmp10 genes was greatly enhanced in PC12-SH2B1beta cells, whereas NGF-induced Plaur and Mmp3 expression was substantially depressed in PC12-SH2B1beta(R555E) cells. Plaur, Mmp3, and Mmp10 are among the 12 genes most highly up-regulated after 6 h of NGF. Their protein products [urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 3 (MMP3), and MMP10] lie in the same pathway of extracellular matrix degradation; uPAR has been shown previously to be critical for NGF-induced neurite outgrowth. Quantitative real-time PCR analysis revealed SH2B1beta enhancement of NGF induction of all three genes and the suppression of NGF induction of all three when endogenous SH2B1 was reduced using short hairpin RNA against SH2B1 and in PC12-SH2B1beta(R555E) cells. NGF-induced levels of uPAR and MMP3/10 and neurite outgrowth through Matrigel (MMP3-dependent) were also increased in PC12-SH2B1beta cells. These results suggest that SH2B1beta stimulates NGF-induced neuronal differentiation at least in part by enhancing expression of a specific subset of NGF-sensitive genes, including Plaur, Mmp3, and/or Mmp10, required for neurite outgrowth.     

Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.     

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.     

Dystrophin Dp71 is required for neurite outgrowth in PC12 cells

To determine the role of Dp71 in neuronal cells, we generated PC12 cell lines in which Dp71 protein levels were controlled by stable transfection with either antisense or sense constructs. Cells expressing the antisense Dp71 RNA (antisense-Dp71 cells) contained reduced amounts of the two endogenous Dp71 isoforms. Antisense-Dp71 cells exhibited a marked suppression of neurite outgrowth upon the induction with NGF or dibutyryl cyclic AMP. Early responses to NGF-induced neuronal differentiation, such as the cessation of cell division and the activation of ERK1/2 proteins, were normal in the antisense-Dp71 cells. On contrary, the induction of MAP2, a late differentiation marker, was disturbed in these cells. Additionally, the deficiency of Dp71 correlated with an altered expression of the dystrophin-associated protein complex (DAPC) members alpha and beta dystrobrevins. Our results indicate that normal expression of Dp71 is essential for neurite outgrowth in PC12 cells and constitute the first direct evidence implicating Dp71 in a neuronal function.     

NGF-induced neurite regeneration is mediated by a ras-independent pathway in PC12 cells.

A rat pheochromocytoma (PC12) cell line (designated MMTV-M17-5) expressing a dominant inhibitory mutant Ha-ras (Ha-ras Asn 17) protein was used to study nerve growth factor (NGF) induced neurite regeneration. Expression of the mutant p21 completely blocked NGF stimulated process formation in these cells. In contrast, neurite outgrowth induced by NGF treatment of primed MMTV-M17-5 cells was not significantly affected by the presence of Ha-ras Asn 17 protein. These observations suggest that, while ras function is required for NGF induced neuronal differentiation of PC12 cells, it is not needed to mediate NGF stimulated neurite regeneration.     

Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

Neurite outgrowth and neuronal differentiation play a crucial role in the development of the nervous system. Understanding of neurotrophins induced neurite outgrowth was important to develop therapeutic strategy for axon regeneration in neurodegenerative diseases as well as after various nerve injuries. It has been reported that extension of neurite and differentiation of sympathetic neuron-like phenotype was modulated by nerve growth factor (NGF) in PC12 cells. In this study, NGF mediated neurite outgrowth was investigated in PC12 cells after liquiritin exposure. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae radix, which is frequently used to treat injury or swelling for its life-enhancing properties as well as detoxification in traditional Oriental medicine. The result showed that liquiritin significantly promotes the neurite outgrowth stimulated by NGF in PC12 cells in dose dependant manners whereas the liquiritin alone did not induce neurite outgrowth. Oligo microarray and RT-PCR analysis further clarified that the neurotrophic effect of liquiritin was related to the overexpression of neural related genes such as neurogenin 3, neurofibromatosis 1, notch gene homolog 2, neuromedin U receptor 2 and neurotrophin 5. Thus, liquiritin may be a good candidate for treatment of various neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease.