Morphological Differentiation Of PC12 Cells Research Articles

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells

Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H2O2 accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.

Neurotrophic factor production in human astrocytoma cells by 2,5,6-tribromogramine via activation of epsilon isoform of protein kinase C

It is known that astrocytes secrete several neurotrophic factors to promote the survival of neurons. For the treatment of neuronal disorders, low molecular weight compounds inducing neurotrophic factor synthesis are useful, because neurotrophic factors are polypeptides which cannot cross the blood brain barrier. When rat pheochromocytoma (PC-12) cells were cultivated in the medium of human astrocytoma cells (1321N1) treated with 2,5,6-tribromogramine, they differentiated to neuron-like cells possessing neurites, indicating that 2,5,6-tribromogramine released neurotrophic factors from 1321N1 cells. In fact, 2,5,6-tribromogramine increased nerve growth factor (NGF) protein synthesis and secretion through mRNA expression. 2,5,6-Tribromogramine inhibited carbachol-induced phosphoinositide hydrolysis as well as phorbol 12,13-myristate acetate did. The inhibition was recovered by bisindolylmaleimide I (GF109203X), a specific protein kinase C (PKC) inhibitor, indicating that 2,5,6-tribromogramine may activate PKC. The morphological differentiation of PC-12 cells by the medium treated with 2,5,6-tribromogramine was also reduced by GF109203X. 2,5,6-Tribromogramine translocated PKC-ɛ but not PKC-α or PKC-ζ, to membrane fraction from cytosol fraction. These results indicate that 2,5,6-tribromogramine promotes the synthesis and secretion of neurotrophic factors including NGF in 1321N1 cells via an activation of PKC-ɛ.

Essential Role for Phospholipase D2 Activation Downstream of ERK MAP Kinase in Nerve Growth Factor-stimulated Neurite Outgrowth from PC12 Cells

The signaling pathway that triggers morphological differentiation of PC12 cells is mediated by extracellular signal-regulated kinase (ERK), the classic mitogen-activated protein (MAP) kinase. However, mediators of the pathway downstream of ERK have not been identified. We show here that phospholipase D2 (PLD2), which generates the pleiotropic signaling lipid phosphatidic acid (PA), links ERK activation to neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. Increased expression of wild type PLD2 (WT-PLD2) dramatically elongated neurites induced by NGF stimulation or transient expression of the active form of MAP kinase-ERK kinase (MEK-CA). The response was activity-dependent, because it was inhibited by pharmacological suppression of the PLD-mediated PA production and by expression of a lipase-deficient PLD2 mutant. Furthermore, PLD2 was activated by MEK-CA, whereas NGF-stimulated PLD2 activation and hypertrophic neurite extension were blocked by an MEK-specific inhibitor. Taken together, these results provide evidence that PLD2 functions as a downstream signaling effector of ERK in the NGF signaling pathway, which leads to neurite outgrowth by PC12 cells.

PC12 cell activation by epidermal growth factor receptor: role of autophosphorylation sites

PC12 cells have been used as a model system for neuronal differentiation due to their ability to alter their phenotype to a sympathetic neuron-like cell in response to nerve growth factor or fibroblast growth factor. Under some conditions, epidermal growth factor (EGF) can also induce PC12 cells to differentiate. To study signaling from the EGF receptor without the confounding effects of endogenous EGF receptors we generated a chimeric receptor comprised of the ectodomain of platelet-derived growth factor (PDGF) receptor in-frame with the transmembrane and cytoplasmic domains of EGF receptor, termed PER. Expression of PER in PC12 cells confers the ability of PDGF to induce differentiation whereas PDGF has no effect on untransfected PC12 cells. This response is kinase activity-dependent since a kinase-deficient mutant (K721M) fails to induce differentiation in response to PDGF. Mutation of five tyrosine residues that are autophosphorylated in response to EGF either individually or in combination had minimal effects on the ability of these receptors to induce morphological PC12 cell differentiation. The PER mutant with all five autophosphorylation sites mutated to phenylalanine (5YF) was equivalently capable of interacting with several important signaling molecules, including Shc, Grb2, Gab1, phospholipase Cγ, and Cbl. Furthermore, both the phosphatidylinositol 3-kinase (PI3K)/Akt and Ras/Erk pathways were activated in a sustained manner when PER or 5YF-expressing cells were stimulated with PDGF. Our results show that the five autophosphorylation sites in the extra-kinase C-terminal domain of EGFR are not required for the ability of EGFR to induce morphological differentiation of PC12 cells.

Lipophilic fraction of Panax ginseng induces neuronal differentiation of PC12 cells and promotes neuronal survival of rat cortical neurons by protein kinase C dependent manner

Panax ginseng is a traditional Chinese herb with a wide range of therapeutic benefits. Recent studies focusing on its effect on the central nervous system have revealed that ginseng has neurotrophic effects including differentiation of neurons. However, most studies involve use of the water-soluble fraction called saponin, and little is known about the effect of the lipophilic fraction. In the present study, we have shown that the lipophilic fraction of ginseng at a concentration of between 0.1 and 50 μg/ml can induce neurite outgrowth of PC12 cells in a dose-dependent manner. Nearly all cells showed morphological differentiation in response to the lipophilic fraction. This morphological differentiation of PC12 cells appeared to be similar to that of NGF. The lipophilic fraction of ginseng also induced neurite extension and promoted survival of rat cortical neurons at a concentration of between 0.025 and 1 μg/ml. These neurotrophic effects on PC12 cells and cortical neurons were not inhibited by K252b, which selectively blocks neurotrophin actions by inhibiting trk-type receptor tyrosine phosphorylation. This suggests that trks do not participate in the neurotrophic action of the lipophilic fraction. However, the effects were completely attenuated by sphingosine, polymyxin B or staurosporin, known inhibitors of protein kinase C (PKC) and calmodulin-dependent kinases. Our results suggest that the lipophilic fraction of ginseng exerts its neurotrophic effects via PKC-dependent pathways.

SH2-B and APS are multimeric adapters that augment TrkA signaling.

Neurotrophins influence growth and survival of sympathetic and sensory neurons through activation of their receptors, Trk receptor tyrosine kinases. Previously, we identified Src homology 2-B (SH2-B) and APS, which are structurally similar adapter proteins, as substrates of Trk kinases. In the present study, we demonstrate that both SH2-B and APS exist in cells as homopentamers and/or heteropentamers, independent of Trk receptor activation. Structure-function analyses revealed that the SH2-B multimerization domain resides within its amino terminus, which is necessary for SH2-B-mediated nerve growth factor (NGF) signaling. Overexpression of SH2-B enhances both the magnitude and duration of TrkA autophosphorylation following exposure of PC12 cells to NGF, and this effect requires the amino-terminal multimerization motif. Moreover, the amino terminus of SH2-B is necessary for TrkA/SH2-B-mediated morphological differentiation of PC12 cells. Together, these results indicate that the multimeric adapters SH2-B and APS influence neurotrophin signaling through direct modulation of Trk receptor autophosphorylation.

Sustained formation of focal adhesions with paxillin in morphological differentiation of PC12 cells.

Differentiation of PC12 cells triggered by nerve growth factor (NGF) is characterized by several well-defined events including induction of a set of neuron-specific genes, gain of membrane excitability, and morphological changes such as neurite outgrowth. Here we report that K252a, a protein kinase inhibitor, converts the proliferation signal of epidermal growth factor (EGF) into the morphological differentiation signal without inducing the sustained activation of ERK and the expression of neurofilament. Major effects of EGF/K252a, found also in the NGF-treated cells, are the sustained mobility shift of paxillin in SDS-PAGE and the promoted association of Crk-II with paxillin. These effects explain the prominent and robust development of peripheral focal adhesion assembly and stress fiber-like structures observed in the early stages of PC12 cell differentiation. These results suggest a model that cytoskeletal reorganization via focal adhesion assembly triggered by NGF provides a signal required for the morphological differentiation of PC12 cells.

The Urokinase Plasminogen Activator Receptor (UPAR) Is Preferentially Induced by Nerve Growth Factor in PC12 Pheochromocytoma Cells and Is Required for NGF-Driven Differentiation

Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.

Sustained Formation of Focal Adhesions with Paxillin in Morphological Differentiation of PC12 Cells

Differentiation of PC12 cells triggered by nerve growth factor (NGF) is characterized by several well-defined events including induction of a set of neuron-specific genes, gain of membrane excitability, and morphological changes such as neurite outgrowth. Here we report that K252a, a protein kinase inhibitor, converts the proliferation signal of epidermal growth factor (EGF) into the morphological differentiation signal without inducing the sustained activation of ERK and the expression of neurofilament. Major effects of EGF/K252a, found also in the NGF-treated cells, are the sustained mobility shift of paxillin in SDS-PAGE and the promoted association of Crk-II with paxillin. These effects explain the prominent and robust development of peripheral focal adhesion assembly and stress fiber-like structures observed in the early stages of PC12 cell differentiation. These results suggest a model that cytoskeletal reorganization via focal adhesion assembly triggered by NGF provides a signal required for the morphological differentiation of PC12 cells.

Bicyclic monoterpene diols induce differentiation of S91 melanoma and PC12 pheochromocytoma cells by a cyclic guanosine-monophosphate-dependent pathway.

Previously, we showed that 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) is an effective inducer of melanogenesis in cultured cells and guinea-pig skin [Brown et al. (1998) J. Invest. Dermatol., 110:428-437]. This study shows that 2,3-cis/exo-pinanediol (2,3-cs/ex-PinD) is a more effective inducer of melanogenesis than 5-NBene-2,2-DM in S91 mouse melanoma cells. Furthermore, 2,3-cs/ex-PinD appears to penetrate guinea-pig skin better than 5-NBene-2,2-DM and to induce higher levels of pigmentation. Both 5-NBene-2,2-DM and 2,3-cs/ex-PinD induce synthesis of nitric oxide (NO) in S91 cells, and the melanogenic activity of both compounds is reduced by inhibitors of the NO/cyclic guanosine monophosphate (cGMP)/protein kinase(PK) G signaling pathway, but not by inhibitors of the PKC or PKA pathways. Thus, these bicyclic monoterpene diols appear to induce melanogenesis by the same pathway in S91 cells as that shown previously for ultraviolet radiation in melanocytes (Romero-Graillet et al. (1996) J. Biol. Chem., 271:28052-28056). These compounds also induce NO synthesis, neurite outgrowth, and tyrosine hydroxylase activity in PC12 pheochromocytoma cells. Neurite outgrowth in PC12 cells is blocked by the guanylate cyclase inhibitor, LY83583 (6-anilino-2,8-quinolinequinone), indicating that, similar to S91 cells, the induction of morphological differentiation of PC12 cells by bicyclic monoterpene diols is regulated by a cGMP-dependent pathway.

Identification and Characterization of Novel Substrates of Trk Receptors in Developing Neurons

Neurotrophins influence growth and survival of specific populations of neurons through activation of Trks, members of the receptor tyrosine kinase (RTK) family. In this report, we describe the identification and characterization of two substrates of Trk kinases, rAPS and SH2-B, which are closely related Src homolog 2 (SH2) domain–containing signaling molecules. rAPS and SH2-B are substrates of TrkB and TrkC in cortical neurons and SH2-B is a substrate of TrkA in sympathetic neurons. Moreover, rAPS and SH2-B bind to Grb2, and both are sufficient to mediate NGF induction of Ras, MAP kinase (MAPK), and morphological differentiation of PC12 cells. Lastly, antibody perturbation and transient transfection experiments indicate that SH2-B, or a closely related molecule, is necessary for NGF-dependent signaling in neonatal sympathetic neurons. Together, these observations indicate that rAPS and SH2-B mediate Trk signaling in developing neurons.

The negative influence of endogenous opioid receptor activity on the differentiation of the rat pheochromocytoma PC12 cells induced by nerve growth factor

We determined the effect of naloxone and morphine on the differentiation of pheochromocytoma cell, the PC12 cells, induced by nerve growth factor (NGF). PC12 cells were grown in medium containing NGF with or without the addition of naloxone or morphine for up to 10-day treatments. NGF-induced morphological differentiation of PC12 cells was manifested by an increase in the percentage of differentiated cells and the average length of neurite per cell. Co-addition of morphine with NGF did not affect both parameters as compared to that of NGF alone. On the contrary, co-addition of naloxone with NGF significantly increased the percentage of differentiated cells, but did not affect the outgrowth of neurites. This effect of naloxone was reversed by the addition of morphine, suggesting that naloxone produced its effect by inhibiting the endogenous activity of opioid receptor. This study indicates a significant functional role of opioid receptor in NGF-induced differentiation of PC12 cells.

Electrically induced neurite outgrowth of PC12 cells on the electrode surface.

Morphological differentiation of PC12 cells cultured on an indium-tin oxide (ITO) electrode has been induced to grow neurites in the absence of nerve growth factor (NGF) by electrical stimulation. Rectangular pulse wave potentials were applied to the electrode at amplitudes of 200 mV and 400 mV with frequencies of 50 Hz, 500 Hz, and 1 kHz. The PC12 cells differentiated most prominently at 200 mV with 100 Hz. No statistically significant differences were observed among the electrically induced neurite lengths. The electrically induced differentiation was completely inhibited by a blockade of calcium influx using LaCl3. This indicates that repeated potential shift in the vicinity of a cellular membrane may stimulate morphological response, probably through calcium ion channels.

Nerve Growth Factor-mediated Activation of the Mitogen-activated Protein (MAP) Kinase Cascade Involves a Signaling Complex Containing B-Raf and HSP90

The nerve growth factor (NGF)-mediated activation of the mitogen-activated protein (MAP) kinase cascade is an obligatory step in the morphological differentiation of PC12 cells. Signal transduction through the MAP kinase cascade is dependent upon activation of p21(ras) which binds directly to Raf family protein kinases, mediating their association with the membrane and activation. PC12 cells express two Raf isoforms, c-Raf and B-Raf. The activation of the MAP kinase cascade in response to NGF is due principally to the action of B-Raf. NGF treatment of PC12 cells resulted in the enhanced phosphorylation of B-Raf and c-Raf, and both exhibit reduced electrophoretic mobilities following stimulation of the cells. The NGF-stimulated phosphorylation of B-Raf was correlated with its enzymatic activation as measured by the phosphorylation of its substrate MEK. However, c-Raf does not exhibit significant levels of activity. B-Raf was present as a component of a high molecular mass complex, which included the molecular chaperone, heat shock protein 90 (HSP90). Importantly, c-Raf did not participate in the formation of such complexes. The B-Raf containing HSP90 complexes were normally present in PC12 cells, and their assembly was not dependent upon NGF stimulation. These data suggest that the ability of B-Raf to activate the MAP kinase cascade is due to its association with a large signaling complex, which is likely to impart signaling pathway specificity.

Nerve growth factor inhibits the expression of long-term potentiation in hippocampal slices

Nerve growth factor and its receptor(s) are present in several parts of the hippocampus (CA1, CA3, dentate gyrus) but nothing is known about their function in this area which plays a fundamental role in learning and memory processes. NGF delivered exogenously to hippocampal slices causes a concentration-dependent, marked reduction in the expression (but not the induction) of long term potentiation (LTP) without altering basal synaptic transmission. The effect is already half maximal at 0.05-0.1 ng ml-1 NGF, is reversible after removal of this growth factor, and is also detectable with a modified version of NGF which has lost its neurite outgrowth promoting activity in PC12 cells. These findings point to a role for hippocampal NGF as a possible modulator of learning and memory processes. Such modulation would be mediated by high-affinity receptors functionally distinct from those promoting morphological differentiation of PC12 cells and other NGF target cells.

Rapid fibroblast growth factor-induced increases in protein phosphorylation and ornithine decarboxylase activity: Regulation by heparin and comparison to nerve growth factor-induced increases

Fibroblast growth factors (FGFs), like nerve growth factor (NGF), induce morphological differentiation of PC12 cells. This activity of FGF is regulated by glycosaminoglycans. To further understand the mechanisms of FGF and glycosaminoglycan actions in PC12 cells, we studied the regulation of protein phosphorylation and ornithine decarboxylase (ODC) activity by FGF in the presence and absence of heparin. As with NGF, aFGF and bFGF increased the incorporation of radioactive phosphate into the protein tyrosine hydroxylase (TH). The increase in TH phosphorylation was localized to the tryptic peptide, T3. Both T3 and T1 phosphorylations occur in response to NGF, but there was no evidence that aFGF or bFGF stimulated the phosphorylation of the T1 peptide. This result suggests differential regulation of second messenger systems by NGF and FGF in PC12 cells. Heparin, at a concentration that potentiated aFGF-induced neurite outgrowth 100-fold (100 μg/ml), did not alter the ability of aFGF to increase S6 phosphorylation or ODC activity. One milligram per milliliter of heparin, a concentration that inhibited bFGF-induced neurite outgrowth, also inhibited bFGF-induced increases in S6 phosphorylation and ODC activity. These observations suggest (i) that acidic and basic FGF activate a protein kinase, possibly protein kinase C, resulting in the phosphorylation of peptide T3 of TH; (ii) that the FGFs and NGF share some but not all second messenger systems; (iii) that heparin potentiates aFGF actions and inhibits bFGF actions in PC12 cells via distinct mechanisms; (iv) that heparin does not potentiate the neurite outgrowth promoting activity of aFGF by enhancing binding to its PC12 cell surface receptor; and (v) that heparin may coordinately regulate several activities of bFGF (induction of protein phosphorylation, ODC and neurite outgrowth) via a common mechanism, most likely by inhibiting the productive binding of bFGF to its PC12 cell surface receptor.

Morphological differentiation of rat pheochromocytoma cells (PC12 cells) by electric stimulation

The effects of electric stimulation on the morphological differentiation of PC12 cells are described. PC12 cells were stimulated with the ‘theta’ (4–7 Hz electroencephalogram (EEG) rhythm) pattern-electric stimulation, which was known to elicit stable long-term potentiation (LTP) in the CA1 region of the hippocampus. The stimulation induced the neurite outgrowth of PC12 cells, as well as nerve growth factor (NGF). This result suggests that the electric signal has a differentiating potential equivalent to the receptor-ligand interaction.

Role of the cAMP‐dependent protein kinase and protein kinase C in regulating the morphological differentiation of PC12 cells

The cell line A126-1B2 is a PC12-derived mutant that is resistant to the toxic effects of dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) and is deficient in adenosine 3':5'-cyclic monophosphate (cAMP)-dependent protein kinase II (PKAII). This mutant formed neurites in response to nerve growth factor (NGF), but not in response to dbcAMP; and dbcAMP did not increase the rate of NGF-dependent neurite formation. Thus, while PKAII is essential for process formation in response to agents that act through the cAMP-dependent pathway, activation of PKAII is not essential for NGF-dependent neurite formation. Unexpectedly, NGF and phorbol 12-myristate 13-acetate (PMA; 10-1,000 nM) synergistically stimulated the formation of short processes that were apparent within 30 min of NGF addition in 85% of these mutant cells. These processes were similar, but not identical, in appearance to the NGF-dependent neurites that formed only after a period of 24-48 hr. This effect is dependent on the activation of protein kinase C (PKC) because an inactive phorbol ester was without effect. In contrast, there was only a small effect of NGF and/or PMA on process formation in wild type cells within the first few hours. The effect of PMA is not augmented by dbcAMP in the A126-1B2 mutant cells. After several hours, PMA caused a concentration-dependent decrease in cell adhesion; and higher concentrations of PMA resulted in a transient detachment of the cells and a loss of neurites. These experiments suggest a role for PKC in the regulation of process formation.

Nerve growth factor induces protein-tyrosine phosphorylation.

When the sympathetic nerve-like cell line PC12 is exposed to nerve growth factor (NGF), there is a rapid and transient phosphorylation of tyrosine residues in cellular proteins, as demonstrated by immunoblotting of cell extracts with high-affinity polyclonal antibodies specific for phosphotyrosine residues. Epidermal growth factor (EGF), which does not cause the morphological differentiation of PC12 cells that is produced by NGF, also induces protein-tyrosine phosphorylation. The methyltransferase inhibitor, 5'-methylthioadenosine, which is known to block the NGF-mediated morphological differentiation of PC12 cells, also inhibits the induction of protein-tyrosine phosphorylation by NGF. 5'-Methylthioadenosine has no effect, however, on EGF-stimulated phosphorylation of tyrosine residues in cellular proteins. In addition, low temperature markedly slows the rate of protein-tyrosine phosphorylation stimulated by NGF, but it has no effect on the time course of protein-tyrosine phosphorylation induced by EGF. These data suggest that NGF and EGF induce protein-tyrosine phosphorylation in PC12 cells by different mechanisms.

Analysis of guanine nucleotide bound to ras protein in PC12 cells

The ras gene product (p21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP-binding proteins, only the GTP-bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p21 (Val-12) but also proto-oncogenic p21(Gly-12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTPγS [(1987) Mol. Cell. Biol. 7, 4553–4556]. In the present report we transformed PC12 cells with the oncogenic ras gene placed under the metallothionein I promoter. It was found that the transformed cells, when induced with Cd 2+, differentiated in the absence of NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p21(Val-12) was mostly present in the GTP-bound form, whereas the endogenous p21(Gly-12) was in the GDP-bound form. These results indicate again that p21·GTP induces the morphological differentiation of PC12 cells.