Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin- deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.     

Grit,a GTPase-activating protein for the Rho family,regulates neurite extension through association with the TrkA receptor and N-Shc and CrkL/Crk adapter molecules

Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.     

Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.     

Neurite outgrowth of mature retinal ganglion cells and PC12 cells requires activity of CK1δ and CK1ε

Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI) enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth.     

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.     

Evidence for Nerve Growth Factor-Potentiating Activities of the Nonpeptidic Compound SR 57746A in PC12 Cells

Abstract: SR 57746A {1-[2-(naphth-2-yl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6-tetrahydropyridine hydrochloride} exhibits neurotrophic activities in vivo and in vitro. We used the rat pheochromocytoma PC12 cell line to investigate in vitro cellular changes induced by SR 57746A. A significant increase in the percentage of cells bearing neurite-like processes was obtained in cells treated by SR 57746A and nerve growth factor (NGF) compared with NGF treatment alone. SR 57746A added alone, however, had no effect on morphogenesis or on survival of cells in serum-free medium. In contrast, SR 57746A induced a "priming" effect on PC12 cells for neurite outgrowth within 6 h of addition of the protein tyrosine kinase inhibitor genistein. An increase in α-actinin content resulted from treatment with SR 57746A. Expression of NGF-mediated acetylcholinesterase and choline acetyltransferase was enhanced within 5 days by SR 57746A. The molecule also induced rapid F-actin redistribution. Within 2 min of incubation, outgrowth of F-actin-containing filopodia was clearly visible at the cell periphery, as previously shown with NGF. It is interesting that this effect of SR 57746A could be mimicked by protein tyrosine kinase inhibitors and abolished by preincubation with sodium orthovanadate, a protein tyrosine phosphatase inhibitor.     

Varicones and Growth Cones: Two Neurite Terminals in PC12 Cells

The rat adrenal pheochromocytoma PC12 cell line is one of the traditional models for the study of neurite outgrowth and growth cone behavior. To clarify to what extent PC12 neurite terminals can be compared to neuronal growth cones, we have analyzed their morphology and protein distribution in fixed PC12 cells by immunocytochemistry. Our results show that that PC12 cells display a special kind of neurite terminal that includes a varicosity in close association with a growth cone. This hybrid terminal, or “varicone”, is characterized by the expression of specific markers not typically present in neuronal growth cones. For example, we show that calpain-2 is a specific marker of varicones and can be detected even before the neurite develops. Our data also shows that a fraction of PC12 neurites end in regular growth cones, which we have compared to hippocampal neurites as a control. We also report the extraordinary incidence of varicones in the literature referred to as “growth cones”. In summary, we provide evidence of two different kinds of neurite terminals in PC12 cells, including a PC12-specific terminal, which implies that care must be taken when using them as a model for neuronal growth cones or neurite outgrowth.     

Mimicry and Inhibition of Nerve Growth Factor Effects: Interactions of Staurosporine, Forskolin, and K252a in PC12 Cells and Normal Rat Chromaffin Cells In Vitro

The structurally similar compounds staurosporine and K252a are potent inhibitors of protein kinases. K252a has previously been reported to inhibit most or all of the effects of nerve growth factor (NGF) on PC12 pheochromocytoma cells, and staurosporine has been reported both to inhibit and to mimic NGF-induced neurite outgrowth from a PC12 cell subclone in a dose-dependent manner. We have studied the interactions of these agents with each other, with NGF, and with forskolin, an activator of adenylate cyclase, on the parent PC12 cell line and on normal neonatal and adult rat chromaffin cells. Staurosporine alone or in conjunction with forskolin induces outgrowth of short neurites from PC12 cells but does not substitute for NGF in promoting cell survival. It does not abolish NGF-induced neurite outgrowth but does reverse the effects of NGF on catecholamine synthesis. K252a abolishes NGF-induced neurite outgrowth but only partially decreases outgrowth induced by NGF plus forskolin. It does not inhibit neurite outgrowth produced by staurosporine or staurosporine plus forskolin. These findings with PC12 cells suggest that staurosporine might act downstream from K252a and NGF on components of one or more signal transduction pathways by which NGF selectively affects the expression of certain traits. Both neonatal and adult rat chromaffin cells show dramatic flattening and extension of filopodia in response to staurosporine, an observation suggesting that some of the same pathways might remain active in cells that do not exhibit a typical NGF response. Only a small amount of neurite outgrowth is observed, however, and only in neonatal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rho‐associated kinase (ROCK) inhibitor,Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.     

Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor,thereby modulating activation of MAP kinase,Akt, and neurite outgrowth in PC12 cells

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.     

KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner

The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3beta (GSK3beta) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3beta inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3beta overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3beta through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3beta and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3beta-dependent manner.     

Dual control of neurite outgrowth by STAT3 and MAP kinase in PC12 cells stimulated with interleukin-6.

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.     

Requirement for specific protein kinase activities during the rapid redistribution of F-actin that precedes the outgrowth of neurites in PC12D cells.

Rapid changes in morphology of PC12D cells, a subline of PC12 cells, in response to various agents were studied in relation to the subsequent outgrowth of neurites. A few minutes after addition of NGF or of dbcAMP, staining of F-actin with rhodamine phalloidin revealed the formation of ruffles around the periphery of cells. Simultaneous relocalization of F-actin to the area of ruffles occurred in response to NGF. A moderate relocalization of F-actin occurred in dbcAMP-treated cells. Other neurite-promoting agents on PC12D cells, such as bFGF, EGF and PMA, also caused ruffling and an identical redistribution of F-actin. The actin bundles then condensed into several dot-like aggregates that subsequently became the growth cones of neurites. When an inhibitor of protein kinase, K-252a, was added, only the NGF-induced morphological change was selectively decreased. By contrast, an inhibitor of protein kinase A, H-89, selectively blocked the dbcAMP-induced change. These are analogous to the effects of those inhibitors on the outgrowth of neurites. These observations indicate that the formation of ruffles with the redistribution of F-actin might be one of the earliest steps in the neurite outgrowth and that the morphological changes might be triggered by the activation of specific protein kinases. Neither cytochalasin B nor colchicine prevented the series of morphological changes. However, processes formed in the presence of cytochalasin B had no filopodium and protrusions formed in the presence of colchicine were shaped like large filopodia. It appears that microtubules and microfilaments may not be absolutely required for the initiation of the rapid morphological changes, but that complete neurites might be formed with contribution by microtubules and by microfilaments.     

Activation of the mitogen-activated protein kinase cascade through nitric oxide synthesis as a mechanism of neuritogenic effect of genipin in PC12h cells

Prominent neurite outgrowth induced by genipin, a plant-derived iridoid, was substantially inhibited by addition of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy-PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH-diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose-dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin-induced neurite outgrowth. Addition of extracellular signal-regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. L-NAME remarkably depressed genipin-stimulated phosphorylation of ERK-1 and -2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These findings suggest that induced NO production followed by cyclic GMP-mediated stimulation of the mitogen-activated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.     

Localization of pp60c-src in growth cone of PC12 cell

By immunocytochemical and biochemical techniques, we observed the localization and expression of pp60c-src in nerve growth factor (NGF)-treated PC12 cells. Immunostaining of pp60c-src is detected in the neuronal soma and the tips of neurites (growth cones). Immunofluorescence in the neurites is less significant. High-resolution microscopy reveals that the location of pp60c-src in growth cone is in good agreement with the adhesive site of growth cone to the substratum. The pp60c-src kinase activity and the pp60c-src protein level increase 3.1- to 3.5-fold and 2.0-fold during differentiation of PC12 cells, respectively. The pp60c-src levels in the neurite fraction are also higher than those in the neuronal soma fraction. These results support the immunocytochemical finding that pp60c-src is localized in growth cones of differentiated PC12 cells. Furthermore, we discuss the possible role of pp60c-src in growth cone.