Identification of T-cadherin as a novel target of DNA methyltransferase 3B and its role in the suppression of nerve growth factor-mediated neurite outgrowth in PC12 cells

Previously we showed that DNA methyltransferase 3b (Dnmt3b) is required for nerve growth factor (NGF)-induced differentiation of PC12 cells to neuronal phenotype. The present study identified T-cadherin (T-Cad) as one of the targets of Dnmt3b by chromatin immunoprecipitation (ChIP) assay. Combined bisulfite restriction analysis and bisulfite sequencing showed that T-Cad promoter was sparsely methylated in PC12 cells. ChIP-CHOP analysis demonstrated that Dnmt3b is associated with T-Cad promoter irrespective of its methylation status. The mRNA and protein levels of T-Cad were markedly elevated in cells depleted of Dnmt3b by antisense or small interfering RNA. Suppression of T-Cad promoter activity by Dnmt3b was independent of its catalytic activity, which was consistent with the insignificant change in T-Cad promoter methylation status in Dnmt3b-depleted cells. In contrast, deletion of its N-terminal ATRX and PWWP domain abolished its repressor function. Association of histone deacetylase 2 (Hdac2) with T-Cad promoter and restoration of the promoter activity from Dnmt3b-mediated suppression upon treatment with Hdac inhibitor indicated involvement of histone deacetylation in this process. NGF-induced neurite outgrowth was inhibited in a dose dependent manner upon ectopic expression of T-Cad in PC12 cells. Immunofluorescence studies showed that T-Cad was redistributed upon NGF treatment, as evident from its concentration in axon growth cones as opposed to its localization at cell-cell contact region in undifferentiated cells. These results demonstrate a novel role of T-Cad in the NGF-mediated differentiation of PC12 cells to neuronal phenotype.     

Overexpression of the trk tyrosine kinase rapidly accelerates nerve growth factor-induced differentiation.

To investigate the role of the gp140trk receptor tyrosine kinase in nerve growth factor (NGF)-induced differentiation, we have overexpressed gp140trk in the NGF-responsive PC12 cell line. Here we demonstrate that overexpression of gp140trk results in marked changes in NGF-induced differentiation. Whereas PC12 cells elaborated neurites after 2 days of continuous exposure to NGF, PC12 cells overexpressing gp140trk by 20-fold(trk-PC12) began this process within hours. Compared with wild-type PC12 cells, trk-PC12 exhibited an increase in both high and low affinity NGF-binding sites. Furthermore, trk-PC12 cells displayed an enhanced level of NGF-dependent gp140trk autophosphorylation, and this activity was sustained for many hours following ligand binding. The tyrosine phosphorylation or activity of several cellular proteins, such as PLC-gamma 1, PI-3 kinase, and Erk1 and the expression of the mRNA for the late response gene transin were also sustained as a consequence of gp140trk overexpression. The data indicate that overexpression of gp140trk in PC12 cells markedly accelerates NGF-induced differentiation pathways, possibly through the elevation of gp140trk tyrosine kinase activity.     

Expression and activity of 2-5A synthetase in the course of differentiation and apoptosis of PC12 cells

The role of IFN-induced 2-5A system in cell differentiation has not been elucidated. While studying differentiation of PC12 cells we found that the simultaneous treatment of cells with NGF and IFN-gamma in serum-containing medium resulted first in the extension of neurites and then apoptosis. On the contrary, in serum-free medium the cells underwent a more rapid neuronal differentiation. Only the doses of NGF which induced the outgrowth of neurites from the cells were able to induce rapid cell death in combined treatment. When the cells were treated subsequently with NGF and IFN-gamma, the induction of death was observed with NGF post-treatment, but not with NGF pretreatment. Relying on these alternative biological responses, we studied the changes in 2-5A synthetase activity and its 43 kDa isoform expression in the course of differentiation and death of PC12 cells. The results of the present work showed that NGF-induced differentiation of the cells did not evoke any increase in 2-5A synthetase activity or any increase in the expression of its 43 kDa isoform. Moreover, the obtained results demonstrated that NGF could not significantly affect the IFN-induced signalling pathway leading to the activation of 2-5A synthetase gene, at least regarding the studied enzyme activity.     

Different Roles for RhoA During Neurite Initiation, Elongation, and Regeneration in PC12 Cells

The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)-induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid-regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites; however, they remained responsive to NGF, as indicated by the activation of extracellular signal-regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were approximately 50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF-induced neuronal differentiation and neurite regeneration.     

Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor-Induced Differentiation of PC12 Cells

Abstract: Phospholipase C γ1 (PLC-γ1) is phosphorylated on treatment of cells with nerve growth factor (NGF). To assess the role of PLC-γ1 in mediating the neuronal differentiation induced by NGF treatment, we established PC12 cells that overexpress whole PLC-γ1 (PLC-γ1PC12), the SH2-SH2-SH3 domain (PLC-γ1SH223PC12), SH2-SH2-deleted mutants (PLC-γ1ΔSH22PC12), and SH3-deleted mutants (PLC-γ1ΔSH3PC12). Overexpressed whole PLC-γ1 or the SH2-SH2-SH3 domain of PLC-γ1 stimulated cell growth and inhibited NGF-induced neurite outgrowth of PC12 cells. However, cells expressing PLC-γ1 lacking the SH2-SH2 domain or the SH3 domain had no effect on NGF-induced neuronal differentiation. Overexpression of intact PLC-γ1 resulted in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2-SH2-SH3 domain of PLC-γ1 did not alter total inositol phosphate accumulation. To investigate whether the SH2-SH2-SH3 domain of PLC-γ1 can mediate the NGF-induced signal, tyrosine phosphorylation of the SH2-SH2-SH3 domain of PLC-γ1 on NGF treatment was examined. The SH2-SH2-SH3 domain of PLC-γ1 as well as intact PLC-γ1 could be tyrosine-phosphorylated on NGF treatment. These results indicate that the overexpressed SH2-SH2-SH3 domain of PLC-γ1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC-γ1 but to the SH2-SH2-SH3 domain of PLC-γ1.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced 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.     

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.     

The adaptor protein SH2B3 (Lnk) negatively regulates neurite outgrowth of PC12 cells and cortical neurons

SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.     

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.     

Thrombopoietin inhibits nerve growth factor-induced neuronal differentiation and ERK signalling

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.     

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.