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

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

The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner

The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.     

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.     

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

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

Spatio-temporal regulation of Rac1 and Cdc42 activity during nerve growth factor-induced neurite outgrowth in PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. It is widely accepted that Rac1 and Cdc42, members of the Rho GTPase family, positively regulate neurite extension through reorganization of the actin cytoskeleton; however, it remains largely unknown when and where Rac1 and Cdc42 are activated during neuritogenesis. This study visualized the spatio-temporal regulation of Rac1 and Cdc42 activities during nerve growth factor (NGF)-induced neurite outgrowth in living PC12 cells by using probes based on the principle of fluorescence resonance energy transfer (FRET). Immediately after the addition of NGF, Rac1 and Cdc42 were transiently activated in broad areas of the cell periphery; a repetitive activation and inactivation cycle was then observed at the motile tips of protrusions. This localized activation, which was more evident in PC12 cells treated with NGF for more than 24 h, might facilitate neurite extension, because the expression of constitutively active mutants of Rac1 and Cdc42 abrogated NGF-induced neurite outgrowth. FRET imaging also delineated a difference between the localization of activated Rac1 and that of Cdc42 within the neurite tips. Experiments with dominant-negative mutants suggested that Rac1 and Cdc42 were activated by a common guanine nucleotide exchange factor(s) in an early stage of the activation phase. Therefore, the difference between Rac1- and Cdc42-activated areas possibly came from the differential localization between Rac1-specific GTPase-activation proteins (GAPs) and Cdc42-specific GAPs. It was concluded that the localized activation of Rac1 and Cdc42 was caused by both guanine nucleotide exchange factors and GAPs, and was important for neurite extension.     

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

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

Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.     

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.     

Binding of Cdc42 to phospholipase D1 is important in neurite outgrowth of neural stem cells

We previously demonstrated that phospholipase D (PLD) expression and PLD activity are upregulated during neuronal differentiation. In the present study, employing neural stem cells from the brain cortex of E14 rat embryos, we investigated the role of Rho family GTPases in PLD activation and in neurite outgrowth of neural stem cells during differentiation. As neuronal differentiation progressed, the expression levels of Cdc42 and RhoA increased. Furthermore, Cdc42 and PLD1 were mainly localized in neurite, whereas RhoA was localized in cytosol. Co-immunoprecipitation revealed that Cdc42 was bound to PLD1 during differentiation, whereas RhoA was associated with PLD1 during both proliferation and differentiation. These results indicate that the association between Cdc42 and PLD1 is related to neuronal differentiation. To examine the effect of Cdc42 on PLD activation and neurite outgrowth, we transfected dominant negative Cdc42 (Cdc42N17) and constitutively active Cdc42 (Cdc42V12) into neural stem cells, respectively. Overexpression of Cdc42N17 decreased both PLD activity and neurite outgrowth, whereas co-transfection with Cdc42N17 and PLD1 restored them. On the other hand, Cdc42V12 increased both PLD activity and neurite outgrowth, suggesting that active state of Cdc42 is important in upregulation of PLD activity which is responsible for the increase of neurite outgrowth.     

Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.     

NGF-dependent formation of ruffles in PC12D cells required a different pathway from that for neurite outgrowth

Two signaling pathways, phosphoinositide 3-kinase (PI-3k)/Akt and Ras/MAPK, are major effectors triggered by nerve growth factor (NGF). Rac1, Cdc42 and GSK-3beta are reported to be targets of PI-3k in the signal transduction for neurite outgrowth. Immediately after NGF was added, broad ruffles were observed temporarily around the periphery of PC12 cells prior to neurite growth. As PC12D cells are characterized by a very rapid extension of neurites in response to various agents, the signaling pathways described above were studied in relation to the NGF-induced formation of ruffles and outgrowth of neurites. Wortmannin, an Akt inhibitor (V), and GSK-3beta inhibitor (SB425286) suppressed the neurite growth in NGF-treated cells, but not in dbcAMP-treated cells. The outgrowth of neurites induced by NGF but not by dbcAMP was inhibited with the expression of mutant Ras. But upon the expression of dominant-negative Rac1, cells often extended protrusions, incomplete neurites, lacking F-actin. Intact neurites were observed in cells with dominant-negative Cdc42. These results suggest that NGF-dependent neurite outgrowth occurs via a mechanism involving activation of the Ras/PI-3K/Akt/GSK-3beta pathway, while dbcAMP-dependent neurite growth might be induced in a distinct manner. However, inhibitors for GSK-3beta and PI-3k (wortmannin) did not suppress the NGF-dependent formation of ruffles. In addition, the formation of ruffles was not inhibited by the expression of mutant Ras. On the other hand, it was suppressed by the expression of dominant-negative Rac1 or Cdc42. These results suggest that the NGF-induced ruffling requires activation of Rac1 and Cdc42, but does not require Ras, PI-3k, Akt and GSK-3beta. Taken together, the NGF-dependent formation of ruffles might not require Ras/PI-3k/Akt/GSK-3beta, but these pathways might contribute to the formation of intact neurites due to combined actions including Rac1.     

Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal-binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin-CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.     

Nerve growth factor stimulates the concentration of TrkA within lipid rafts and extracellular signal-regulated kinase activation through c-Cbl-associated protein

Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.     

The Human Rho-GEF trio and its target GTPase RhoG are involved in the NGF pathway, leading to neurite outgrowth

Rho-GTPases control a wide range of physiological processes by regulating actin cytoskeleton dynamics. Numerous studies on neuronal cell lines have established that Rac, Cdc42, and RhoG activate neurite extension, while RhoA mediates neurite retraction. Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by accelerating GDP/GTP exchange. Trio displays two Rho-GEF domains, GEFD1, activating the Rac pathway via RhoG, and GEFD2, acting on RhoA, and contains numerous signaling motifs whose contribution to Trio function has not yet been investigated. Genetic analyses in Drosophila and in Caenorhabditis elegans indicate that Trio is involved in axon guidance and cell motility via a GEFD1-dependent process, suggesting that the activity of its Rho-GEFs is strictly regulated. Here, we show that human Trio induces neurite outgrowth in PC12 cells in a GEFD1-dependent manner. Interestingly, the spectrin repeats and the SH3-1 domain of Trio are essential for GEFD1-mediated neurite outgrowth, revealing an unexpected role for these motifs in Trio function. Moreover, we demonstrate that Trio-induced neurite outgrowth is mediated by the GEFD1-dependent activation of RhoG, previously shown to be part of the NGF (nerve growth factor) pathway. The expression of different Trio mutants interferes with NGF-induced neurite outgrowth, suggesting that Trio may be an upstream regulator of RhoG in this pathway. In addition, we show that Trio protein accumulates under NGF stimulation. Thus, Trio is the first identified Rho-GEF involved in the NGF-differentiation signaling.     

Sprouty4 is an endogenous negative modulator of TrkA signaling and neuronal differentiation induced by NGF

The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.     

JNK phosphorylation of paxillin, acting through the Rac1 and Cdc42 signaling cascade, mediates neurite extension in N1E-115 cells

Neurons extend neurites from the cell body before formation of the polarized processes of an axon and dendrites. Neurite outgrowth involves remodeling of the cytoskeletal components, which are initially regulated by small GTPases of the Rho family. Here we show that c-Jun N-terminal kinase (JNK), which is controlled by Rho GTPases Rac1 and Cdc42, is activated following neurite extension in mouse N1E-115 neuroblastoma cells as a model. The extension is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) and Clostridium difficile Toxin B, the inhibitor for Rho GTPases. Additionally, paxillin, the multifunctional focal adhesion protein, is phosphorylated at Ser 178 by upregulation of the Rac1/Cdc42/JNK cascade. Conversely, transfection of the paxillin construct harboring the Ser 178-to-Ala mutation into cells inhibits neurite extension. Taken together, these results suggest the novel role of the Rac1/Cdc42/JNK signaling cascade in neurite extension and indicate that the downstream target paxillin may be one of the convergent points of various signaling pathways underlying neurite extension.