Protein tyrosine phosphatase activation during nerve growth factor-induced neuronal differentiation of PC12 cells.

We have studied the role of protein tyrosine phosphatases (PTPases) during neuronal differentiation of PC12 cells. Nerve growth factor (NGF), a well-characterized differentiating agent for these cells, led to a decrease in DNA synthesis within 24 h. This was accompanied by a 2- to 3-fold increase in the activity of PTPases, measured as the dephosphorylation of polyacidic or polybasic substrates phosphorylated on tyrosine. PTPase activation was independent of cell density and proportional to NGF concentration, with a half-maximal effect occurring at 0.35 nM. High-performance liquid chromatography size exclusion chromatography revealed that PTPases with molecular masses of 550, 300, and 60 kilodaltons were activated in response to NGF. Additional studies showed that the presence of NGF made PC12 cells refractory to the mitogenic effect of epidermal growth factor. Our data indicate that NGF-induced neuronal differentiation and growth arrest in PC12 cells are associated with activation of several PTPases. We speculate that PTPase activation in response to NGF may inhibit the mitogenic actions of other growth factors.     

Protein deacetylase SIRT1 in the cytoplasm promotes nerve growth factor-induced neurite outgrowth in PC12 cells

SIRT1, a NAD⁺-dependent protein deacetylase, is known to have neural functions. However, despite its cytoplasmic expression in some neural cells, its cytoplasmic function, if any, is unknown. Here we found that PC12 (pheochromocytoma) cells expressed SIRT1 in the cytoplasm. Nerve growth factor (NGF)-induced neurite outgrowth of these cells was promoted by activators of SIRT1, while inhibitors of SIRT1 or SIRT1-siRNA significantly inhibited it. The overexpression of a mutant SIRT1 that localised to the cytoplasm but not the nucleus enhanced the NGF-dependent neurite outgrowth, and a cytoplasmic dominant-negative SIRT1 suppressed it. Thus, cytoplasmic SIRT1 increases the NGF-induced neurite outgrowth of PC12 cells.     

Determining the oxidation states of manganese in PC12 and nerve growth factor-induced PC12 cells

Excessive brain Mn can produce toxicity with symptoms resembling parkinsonism. This syndrome, called "manganism," correlates with loss of dopamine in the striatum and cell death in the striatum and globus pallidus. A common hypothesis is that cell damage in Mn toxicity is caused by oxidation of important cell components by Mn3+. Determination of the amount of Mn3+ present, under a range of conditions, in neuronal cells and brain mitochondria represents an important step in evaluating the "damage through oxidation by Mn3+ hypothesis." In an earlier paper we used X-ray absorption near-edge structure (XANES) spectroscopy to determine the amount of Mn2+ and Mn3+ in brain mitochondria under a range of conditions. Here we extend the study to investigate the evidence for formation of Mn3+ through oxidation of Mn2+ by ROS in PC12 cells and in PC12 cells induced with nerve growth factor (NGF) to display a phenotype more like that of neurons. Although the results suggest that very small amounts of Mn3+ might be present at low Mn levels, probably in Mn superoxide dismutase, Mn3+ is not stabilized by complex formation in these cells and therefore does not accumulate to detectable amounts.     

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.     

PC12 cells express juvenile microtubule-associated proteins during nerve growth factor-induced neurite outgrowth

Microtubule-associated proteins (MAPs) are believed to play an important role in regulating the growth of neuronal processes. The nerve growth factor-induced differentiation of PC12 pheochromocytoma cells is a widely used tissue culture model for studying this mechanism. We have found that contrary to previous suggestions, the major MAPs of adult brain, MAP1 and MAP2, are minor components of PC12 cells. Instead two novel MAPs characteristic of developing brain, MAP3 and MAP5, are present and increase more than 10-fold after nerve growth factor treatment; the timing of these increases coinciding with the bundling of microtubules and neurite outgrowth. Immunocytochemical staining showed that MAP3 and MAP5 are initially distributed throughout the cytoplasm. Subsequently MAP5 becomes associated with microtubules in both neurites and growth cones but MAP3 distribution remained diffuse. Thus MAP3 and MAP5, which are characteristic of developing neurons in the juvenile brain, are also induced in PC12 cells during neurite outgrowth in culture. In contrast MAP1, which is characteristic of mature neurons, does not increase during PC12 cell differentiation. These results provide evidence that one set of MAPs is expressed during neurite outgrowth and a different set during the maintenance of neuronal form. It also appears that the PC12 system is an appropriate model for studying the active neurite growth phase of neuronal differentiation but not for neuronal maturation.     

Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells

Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. We have analysed the effect of neuroserpin on growth factor-induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.     

Effects of fluvoxamine on nerve growth factor-induced neurite outgrowth inhibition by dexamethasone in PC12 cells

In the present study, we examined the effects of fluvoxamine on nerve growth factor (NGF)-induced neurite outgrowth inhibition by dexamethasone (DEX) in PC12 cells. Fluvoxamine increased NGF-induced neurite outgrowth. Compared with co-treatment with NGF and fluvoxamine, p-Akt levels were higher than the values without fluvoxamine. The phosphorylated extracellular regulated kinase 1/2 levels were slightly increased by co-treatment with NGF and fluvoxamine. Fluvoxamine concentration-dependently improved NGF-induced neurite outgrowth inhibition by DEX. Fluvoxamine also improved the decrease in the NGF-induced p-Akt level caused by DEX. Interestingly, the sigma-1 receptor antagonist NE-100 blocked the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX. The selective sigma-1 receptor agonist PRE-084 also improved NGF-induced neurite outgrowth inhibition by DEX, which is blocked by NE-100. These results indicate that the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX may be attributable to the phosphorylation of Akt and the sigma-1 receptor.     

Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.     

Phosphorylation of stathmin and other proteins related to nerve growth factor-induced regulation of PC12 cells

We previously identified a set of soluble proteins whose phosphorylation could be originally related to the multihormonal regulations of anterior pituitary cells. Among these proteins, stathmin (proteins 7 and 8) was found to be ubiquitous and mostly abundant in neurons. Interestingly, stathmin and some other phosphoproteins of the same set could be identified also in PC12 cells in culture. Their phosphorylation was stimulated in these cells by nerve growth factor (NGF) in a way associated with its short term actions, probably corresponding to the early steps of its neuronal differentiating activity. In addition, the same proteins had their phosphorylation stimulated in the presence of fibroblast growth factor, known to stimulate PC12 cell differentiation in a way similar to NGF. A pharmacological analysis allowed us to distinguish three characteristic subsets of phosphoproteins, respectively, affected by cAMP-dependent agents, by cAMP-independent ones, or by both types of agents. Moreover, phosphorylation of stathmin and some other proteins was additive in the presence of NGF and of the cAMP-promoting agent forskolin. Altogether, the present results unravel some intracellular mechanisms related to the regulation of PC12 cells by extracellular effectors. They extend to the regulation of cell differentiation in our recent model for stathmin (Sobel, A., Boutterin, M-C., Beretta, L., Chneiweiss, H., Doye, V., and Peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772) as an ubiquitous intracellular relay possibly integrating the actions of diverse second messenger pathways involved in cell regulations.     

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.     

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells

All transforming growth factor beta (TGF-beta) superfamily members are synthesized as precursors with prodomain sequences that are proteolytically removed by subtilisin-like proprotein convertases (SPCs). For most superfamily members, this is believed sufficient for activation. Exceptions are TGF-betas 1 to 3 and growth differentiation factor 8 (GDF8), also known as myostatin, which form noncovalent, latent complexes with their SPC-cleaved prodomains. Sequence similarities between TGF-betas 1 to 3, myostatin, and superfamily member GDF11, also known as bone morphogenetic protein 11 (BMP11), prompted us to examine whether GDF11 might be capable of forming a latent complex with its cleaved prodomain. Here we demonstrate that GDF11 forms a noncovalent latent complex with its SPC-cleaved prodomain and that this latent complex is activated via cleavage at a single specific site by members of the developmentally important BMP1/Tolloid family of metalloproteinases. Evidence is provided for a molecular model whereby formation and activation of this complex may play a general role in modulating neural differentiation. In particular, mutant GDF11 prodomains impervious to cleavage by BMP1/Tolloid proteinases are shown to be potent stimulators of neurodifferentiation, with potential for therapeutic applications.     

Potentiation of nerve growth factor-induced neurite outgrowth in PC12 cells by a Coptidis Rhizoma extract and protoberberine alkaloids

A methanol extract of Coptidis Rhizoma effectively enhanced the outgrowth of neurite in PC12 cells induced by nerve growth factor (NGF). Following solvent partition and preparative HPLC, berberine was isolated as the major active compound. Berberine enhanced the proportion of neurite-bearing cells in a dose-dependent manner without cytotoxicity. Its structural relatives, palmatine and coptisine, showed a slightly weaker NGF-enhancing effect than berberine. These three alkaloids inhibited acetylcholinesterase activity at a level comparable to that of physostigmine, but this inhibition was not responsible for the potentiation of NGF-induced neurite outgrowth. It is demonstrated for the first time that protoberberine alkaloids potentiated the NGF-induced differentiation of neural cells.     

Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells

Nitric oxide (NO) is a signaling molecule implicated in a spectrum of cellular processes including neuronal differentiation. The signaling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase and S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. However, little is known about the mechanisms involved and the target proteins for endogenous NO during the progression of neuronal differentiation. To address this question, we investigated the presence, localization, and subcellular distribution of nitrated proteins during neurotrophin-induced differentiation of PC12 cells. We find that some proteins show basal levels of tyrosine nitration in PC12 cells grown in the absence of nerve growth factor (NGF) and that nitration levels increase significantly after 2 days of incubation with this neurotrophin. Nitrated proteins accumulate over a period of several days in the presence of NGF. We demonstrate that this nitration is coupled to activation of nitric oxide synthase. The subcellular distribution of nitrated proteins changes during PC12 cell differentiation, displaying a shift from the cytosolic to the cytoskeletal fraction and we identified alpha-tubulin as the major target of nitration in PC12 cells by N-terminal sequence and MALDI-TOF analyses. We conclude that tyrosine nitration of proteins could be a novel molecular mechanism involved in the signaling pathway by which NO modulates NGF-induced differentiation in PC12 cells.     

Potentiation of nerve growth factor-induced neurite outgrowth in PC12 cells by ifenprodil: the role of sigma-1 and IP3 receptors

In addition to both the α1 adrenergic receptor and N-methyl-D-aspartate (NMDA) receptor antagonists, ifenprodil binds to the sigma receptor subtypes 1 and 2. In this study, we examined the effects of ifenprodil on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Ifenprodil significantly potentiated NGF-induced neurite outgrowth, in a concentration-dependent manner. In contrast, the α1 adrenergic receptor antagonist, prazosin and the NMDA receptor NR2B antagonist, Ro 25-6981 did not alter NGF-induced neurite outgrowth. Potentiation of NGF-induced neurite outgrowth mediated by ifenprodil was significantly antagonized by co-administration of the selective sigma-1 receptor antagonist, NE-100, but not the sigma-2 receptor antagonist, SM-21. Similarly, ifenprodil enhanced NGF-induced neurite outgrowth was again significantly reduced by the inositol 1,4,5-triphosphate (IP(3)) receptor antagonists, xestospongin C and 2-aminoethoxydiphenyl borate (2-APB) treatment. Furthermore, BAPTA-AM, a chelator of intracellular Ca(2+), blocked the effects of ifenprodil on NGF-induced neurite outgrowth, indicating the role of intracellular Ca(2+) in the neurite outgrowth. These findings suggest that activation at sigma-1 receptors and subsequent interaction with IP(3) receptors may mediate the pharmacological effects of ifenprodil on neurite outgrowth.     

The ras suppressor, RSU-1, enhances nerve growth factor-induced differentiation of PC12 cells and induces p21CIP expression.

The Rsu-1 Ras suppressor gene was isolated based on its ability to inhibit v-Ras transformation. Using Rsu-1 transfectants of the pheochromocytoma cell line PC12, we demonstrated previously that Rsu-1 expression inhibited Jun kinase activation but enhanced Erk2 activation in response to epidermal growth factor. In the present study, the Rsu-1 PC12 transfectants were used to investigate the role of Rsu-1 in nerve growth factor (NGF)- and v-Ki-ras-mediated neuronal differentiation. NGF-induced neurite extension was enhanced, not inhibited, by the expression of Rsu-1 in PC12 cells. The activation of Erk kinase activity in response to NGF was sustained longer in the Rsu-1 transfectants compared with the vector control cells. During NGF-mediated differentiation, an increase in the expression of specific mRNAs for the early response genes Fos, cJun, and NGF1a was detected in both the vector control and Rsu-1 transfectants. The expression of the differentiation-specific genes VGF8 and SCG10 was similar in Rsu-1 transfectants compared with the vector control cells. The induction of Rsu-1 expression in these cell lines did not inhibit v-Ki-ras-induced differentiation, as measured by neurite extension. These data suggest that although Rsu-1 blocked some Ras-dependent response(s), these responses were not required for differentiation. Moreover, the induction of Rsu-1 expression in the PC12 clones resulted in growth inhibition and p21(WAF/CIP) expression. Hence, Rsu-1 expression enhances NGF-induced differentiation while inhibiting the growth of cells.     

Nerve growth factor-induced decrease in the calpain activity of PC12 cells

PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. Treatment with nerve growth factor causes the cells to differentiate. One of the hallmarks of this differentiation is the generation of neurites. PC12 cells contain both calpain I and calpain II; about 90% of the total calpain activity is due to calpain II. Treatment of the cells with nerve growth factor causes a time-dependent decrease in calpain activity, more than 50% being lost over a 5-day period. Both the decrease in calpain activity and the growth of neurites are reversible upon the removal of nerve growth factor from the cultures. Agents other than nerve growth factor that cause neurite outgrowth, such as fibroblast growth factor and dibutyryl cyclic AMP, also cause a decrease in calpain activity. Calpain levels, as detected with immunoblotting or immunohistochemistry, show no decrease. Removal of calpastatin, the endogenous inhibitor of the calpains, by phenyl-Sepharose chromatography increases the calpain activity of extracts from both control and nerve growth factor-treated cells and brings the activity in the extracts from treated cells up to the activity in those from controls. Calpastatin-containing fractions from extracts of nerve growth factor-treated cells inhibit more calpain activity than do comparable fractions from control cells. These studies suggest that nerve growth factor causes a decrease in the activity of calpain in morphologically differentiating PC12 cells by causing an increase in the activity of calpastatin.