A role for the SHP-2 tyrosine phosphatase in nerve growth-induced PC12 cell differentiation.

SHP-1 and SHP-2 are intracellular protein tyrosine phosphatases containing two adjacent src homology 2 domains that target these phosphatases to cell surface receptor signaling complexes and play a role in receptor signal transduction. In this report the PC12 cell system was used to investigate the potential roles of SHP-1 and SHP-2 in the induction of neuronal differentiation by nerve growth factor (NGF). By using neurite outgrowth as a marker for differentiation, the effects of transfected constructs of SHP-1 and SHP-2 were assessed. Overexpression of a catalytically inactive SHP-2, but not a catalytically inactive SHP-1, blocked NGF-stimulated neurite outgrowth. The mitogen-activated protein kinase (MAPK) signaling cascade is important for the morphological differentiation in PC12 cells, and both SHP-1 and SHP-2 have been implicated to act upstream of MAPK in other receptor signaling systems. A positive role for SHP-2 but not SHP-1 in the activation of MAPK by NGF was demonstrated by introduction of the SHP-2 phosphatase mutants along with hemagglutinin-tagged MAPK. Coexpression studies with the SHP-2 mutant along with mutant forms of MAPK kinase suggested that SHP-2 functions upstream of MAPK kinase and MAPK in NGF-induced neurite outgrowth.     

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.     

Nerve growth factor signals through TrkA,phosphatidylinositol 3-kinase,and Rac1 to inactivate RhoA during the initiation of neuronal differentiation of PC12 cells

In PC12 rat pheochromocytoma cells, nerve growth factor (NGF)-induced neuronal differentiation is blocked by constitutively active dominant mutants of RhoA but augmented by negative ones, suggesting a not yet elucidated inhibitory signaling link between NGF receptors and RhoA. Here we show that NGF treatment rapidly translocates RhoA from the plasma membrane to the cytosol and simultaneously decreases RhoA affinity to its target Rho-associated kinase (ROK), a key mediator of neurite outgrowth. This effect was transient, because after 2 days of NGF treatment, RhoA relocated from the cytosol to the plasma membrane, and its GTP loading returned to a level found in undifferentiated cells. Inhibition of RhoA is mediated by activation of the TrkA receptor, because NGF failed to induce RhoA translocation and inhibition of ROK binding in nnr5 cells that lack TrkA, whereas the inhibition was reconstituted in receptor add-back B5 cells. In MM17-26 cells, which due to expression of dominant negative Ras do not differentiate, NGF-stimulated transient RhoA inhibition was unaffected. The inhibitory pathway from TrkA to RhoA involves phosphatidylinositol-3-kinase (PI3K), because the inhibitors LY294002 or wortmannin prevented NGF-induced RhoA translocation and increased RhoA association with ROK. Furthermore, inhibition of PI3K significantly reduced NGF- mediated Rac1 activation, whereas dominant negative Rac1 abolished the inhibitory signaling to RhoA. Taken together, these data indicate that NGF-mediated activation of TrkA receptor stimulates PI3K, which in turn increases Rac1 activity to induce transient RhoA inactivation during the initial phase of neurite outgrowth.     

Overexpression of the protein phosphatase 2A regulatory subunit Bgamma promotes neuronal differentiation by activating the MAP kinase (MAPK) cascade

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional regulator of cellular signaling. Variable regulatory subunits associate with a core dimer of scaffolding and catalytic subunits and are postulated to dictate substrate specificity and subcellular location of the heterotrimeric PP2A holoenzyme. The role of brain-specific regulatory subunits in neuronal differentiation and signaling was investigated in the PC6-3 subline of PC12 cells. Endogenous Bbeta, Bgamma, and B'beta protein expression was induced during nerve growth factor (NGF)-mediated neuronal differentiation. Transient expression of Bgamma, but not other PP2A regulatory subunits, facilitated neurite outgrowth in the absence and presence of NGF. Tetracycline-inducible expression of Bgamma caused growth arrest and neurofilament expression, further evidence that PP2A/Bgamma can promote differentiation. In PC6-3 cells, but not non-neuronal cell lines, Bgamma specifically promoted long lasting activation of the mitogen-activated protein (MAP) kinase cascade, a key mediator of neuronal differentiation. Pharmacological and dominant-negative inhibition and kinase assays indicate that Bgamma promotes neuritogenesis by stimulating the MAP kinase cascade downstream of the TrkA NGF receptor but upstream or at the level of the B-Raf kinase. Mutational analyses demonstrate that the divergent N terminus is critical for Bgamma activity. These studies implicate PP2A/Bgamma as a positive regulator of MAP kinase signaling in neurons.     

Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.     

Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells

We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF. Rsk1 and Rsk2 mutated in the PDK1-binding site were not activated by either NGF or MEK1-DD. Expression of constitutively active Rsk1 or Rsk2 in PC12 cells resulted in highly active proteins whose levels of activity did not change either with NGF treatment or after coexpression with MEK1-DD. Rsk2-CA expression had no detectable effect on the cells. However, expression of Rsk1-CA led to differentiation of PC12 cells even in the absence of NGF, as evidenced by neurite outgrowth. Differentiation was not observed with a nonactive Rsk1-CA that was mutated in the PDK1-binding site. Expression of Rsk1-CA did not lead to activation of the endogenous MAPK pathway, indicating that Rsk1 is sufficient to induce neurite outgrowth and is the only target of MAPK required for this effect. Collectively, our data demonstrate a key role for Rsk1 in the differentiation process of PC12 cells.     

Biochemical and biological characterization of a neuroendocrine-associated phosphatase

The biochemical and biological properties of a novel neuroendocrine-associated phosphatase (NEAP) were characterized. NEAP had a sequence characteristic of a dual-specificity phosphatase (DSP), and was preferentially expressed in neuroendocrine cells/tissues as well as in skeletal muscle and heart. Expression of NEAP was up-regulated in nerve growth factor (NGF)-treated, differentiated PC12 cells. NEAP was cytosolic and did not apparently have effects against extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase activated by various stimuli. Although NEAP and MAPK phosphatase (MPK)-1 showed similar phosphatase activity towards p-nitro phenylphosphate (pNPP), in contrast to MKP-1, NEAP did not dephosphorylate JNK and p38-MAPK in vitro. Overexpression of NEAP, but not the C152S mutant, in PC12 cells suppressed NGF-induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt activation. Overexpression of NEAP also suppressed neurite outgrowth induced by NGF and sensitized PC12 cells to cisplatin-induced apoptosis. Suppression of NEAP by RNA interference enhanced NGF-induced neurite outgrowth and Akt activation. Our results indicated that, unlike other DSPs, down-regulation of conventional MAPKs was not the major function of NEAP. Furthermore, NEAP might be involved in neuronal differentiation via regulation of the PI3K/Akt signaling.     

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.     

Subtype-specific neuronal differentiation of PC12 cells transfected with alpha2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.     

Rin GTPase couples nerve growth factor signaling to p38 and b-Raf/ERK pathways to promote neuronal differentiation

In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development. Rin is expressed exclusively in neurons and is activated by neurotrophin signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to nerve growth factor (NGF)-mediated neuronal differentiation. Most surprisingly, although Rin was unable to stimulate MAP kinase activity in NIH 3T3 cells, it potently activated isoform-specific p38alpha MAP kinase signaling and weakly stimulated ERK signaling in pheochromocytoma (PC6) cells. This cell-type specificity is explained in part by the finding that Rin binds and stimulates b-Raf but does not activate c-Raf. Accordingly, selective down-regulation of Rin in PC6 cells suppressed neurotrophin-elicited activation of b-Raf and p38, without obvious effects on NGF-induced ERK activation. Moreover, the ability of NGF to promote neurite outgrowth was inhibited by Rin knockdown. Together, these observations establish Rin as a neuronal specific regulator of neurotrophin signaling, required to couple NGF stimulation to sustain activation of p38 MAP kinase and b-Raf signaling cascades required for neuronal development.     

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.     

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.     

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.     

RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.     

The phosphoprotein stathmin is essential for nerve growth factor- stimulated differentiation

Stathmin is a ubiquitous cytosolic protein which undergoes extensive phosphorylation in response to a variety of external signals. It is highly abundant in developing neurons. The use of antisense oligonucleotides which selectively block stathmin expression has allowed us to study directly its role in rat PC12 cells. We show that stathmin depletion prevents nerve growth factor (NGF)-stimulated differentiation of PC12 cells into sympathetic-like neurons although the expression of several NGF-inducible genes was not affected. Furthermore, we found that stathmin phosphorylation in PC12 cells which is induced by NGF depends on mitogen-activated protein kinase (MAPK) activity. We conclude that stathmin is an essential component of the NGF-induced MAPK signaling pathway and performs a key role during differentiation of developing neurons.     

Serine phosphorylation differentially affects RhoA binding to effectors: implications to NGF-induced neurite outgrowth

Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.