A possible involvement of cytoplasmic Ca2+ in sodium dependency of neurite outgrowth of rat pheochromocytoma PC12 cells

The effects of extracellular Na⁺, K⁺ and Cl^? on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na⁺ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60–150 mM Na⁺, whereas it decreased significantly at lower concentrations of Na⁺. Na⁺ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast, neurite outgrowth was independent of K⁺ in the range 5–106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl^? replaced by NO^?₃, SO^2?₄, or 2-hydroxyethanesulfonate. Thus, extracellular Na⁺ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca²⁺ concentration monitored by a fluorescent dye sensitive to Ca²⁺.     

Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

Neurite outgrowth and neuronal differentiation play a crucial role in the development of the nervous system. Understanding of neurotrophins induced neurite outgrowth was important to develop therapeutic strategy for axon regeneration in neurodegenerative diseases as well as after various nerve injuries. It has been reported that extension of neurite and differentiation of sympathetic neuron-like phenotype was modulated by nerve growth factor (NGF) in PC12 cells. In this study, NGF mediated neurite outgrowth was investigated in PC12 cells after liquiritin exposure. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae radix, which is frequently used to treat injury or swelling for its life-enhancing properties as well as detoxification in traditional Oriental medicine. The result showed that liquiritin significantly promotes the neurite outgrowth stimulated by NGF in PC12 cells in dose dependant manners whereas the liquiritin alone did not induce neurite outgrowth. Oligo microarray and RT-PCR analysis further clarified that the neurotrophic effect of liquiritin was related to the overexpression of neural related genes such as neurogenin 3, neurofibromatosis 1, notch gene homolog 2, neuromedin U receptor 2 and neurotrophin 5. Thus, liquiritin may be a good candidate for treatment of various neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease.     

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1-TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75(NTR)-IKK(2)-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK(2) dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent "molecular damper" maintaining a submaximal velocity of neurite extension.     

Immunosuppressant FK506 induces sustained activation of MAP kinase and promotes neurite outgrowth in PC12 mutant cells incapable of differentiating

During the continuous culturing of neural PC12 cells, a drug hypersensitive PC12 mutant cell line (PC12m3) was obtained, which demonstrated high neurite outgrowth when stimulated by various drugs. When the immunosuppressant drug FK506 and nerve growth factor (NGF) were introduced to the PC12m3 cells, the frequency of neurite outgrowth increased approximately 40-fold for NGF alone. However, the effect of FK506 on neuritogenesis in PC12 parental and drug insensitive PC12m1 mutant cells was much lower than in PC12m3 cells. The sustained activation of mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth of PC12 cells. Interestingly, the drug hypersensitive PC12m3 cells exhibited the sustained activation of MAP kinase with FK506 in comparison to low or no activities in PC12 parental or drug insensitive PC12m1 cells. These results indicate that PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Opposite regulatory effects of TRPC1 and TRPC5 on neurite outgrowth in PC12 cells

The transient receptor potential (TRPC) family of Ca^2 + permeable, non-selective cation channels is abundantly expressed in the brain, and can function as store-operated (SOC) and store-independent channels depending on their interaction with the ER Ca^2 + sensor STIM1. TRPC1 and TRPC5 have critical roles in neurite outgrowth, however which of their functions regulate neurite outgrowth is unknown. In this study, we investigated the effects of TRPC channels and their STIM1-induced SOC activity on neurite outgrowth of PC12 cells. We report that PC12 cell differentiation down-regulates TRPC5 expression, whereas TRPC1 expression is retained. TRPC1 and TRPC5 interact with STIM1 through the STIM1 ERM domain. Transfection of TRPC1 and TRPC5 increased the receptor-activated Ca^2 + influx that was markedly augmented by the co-expression of STIM1. Topical expression of TRPC1 in PC12 cells markedly increased neurite outgrowth while that of TRPC5 suppressed neurite outgrowth. Suppression of neurite outgrowth by TRPC5 requires the channel function of TRPC5. However, strikingly, multiple lines of evidence show that the TRPC1-induced neurite outgrowth was independent of TRPC1-mediated Ca^2 + influx. Thus, a) TRPC1 and TRPC5 similarly increased Ca^2 + influx but only TRPC1 induced neurite outgrowth, b) the constitutively STIM1^D76A mutant that activates Ca^2 + influx by TRPC and Orai channels did not increase neurite outgrowth, c) co-expression of TRPC5 with TRPC1 suppressed the effect of TRPC1 on neurite outgrowth, d) and most notable, channel-dead pore mutant of TRPC1 increased neurite outgrowth to the same extent as TRPC1^WT. Suppression of TRPC1-induced neurite outgrowth by TRPC5 was due to a marked reduction in the surface expression of TRPC1. We conclude that the regulation of neurite outgrowth by TRPC1 is independent of Ca^2 + influx and TRPC1-promoted neurite outgrowth depends on the surface expression of TRPC1. It is likely that TRPC1 acts as a scaffold at the cell surface to assemble a signaling complex to stimulate neurite outgrowth.     

Dual control of neurite outgrowth by STAT3 and MAP kinase in PC12 cells stimulated with interleukin-6.

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.     

Electrical stimulation promotes nerve growth factor-induced neurite outgrowth and signaling

Background

Neurotrophins are important regulators for neural development and regeneration. Nerve growth factor (NGF) therapy has been tested in various models of neural injury and degeneration. However, whether NGF can reach target tissues and maintain effective concentration for a certain period of time remains uncertain. To facilitate neural regeneration, we investigate the possibility of combining NGF and electrical stimulation (ES) in promoting neurite outgrowth, an essential process during neural regeneration.

Methods

PC12 cells were seeded on collagen and indium tin oxide (ITO)-coated area on the transparent conductive devices. Cells were then subjected to the combination of ES and NGF treatment. Neurite outgrowth was compared.

Results

Our findings suggest that ES of 100 mV/mm together with NGF provides optimal effect on neurite outgrowth of PC12 cells. ES increases NGF-induced neurite length but reduces neurite branching, indicative of its primary effect on neurite elongation instead of initiation. One mechanism that ES enhances neurite outgrowth is through increasing NGF-induced phosphorylation of ERK1/2 (pERK1/2) and expression of Egr1 gene. ES has previously been demonstrated to increase the activity of protein kinase C (PKC). Our result indicates that activating PKC further increases NGF-induced pERK1/2 and thus neurite outgrowth.

Conclusion

It is likely that ES promotes NGF-induced neurite outgrowth through modulating the activity of ERK1/2.

General significance

Findings from this study suggest that combining ES and NGF provides a promising strategy for promoting neurite outgrowth.     

ErbB-4 activation promotes neurite outgrowth in PC12 cells

Neu differentiation factor (NDF; also known as neuregulin) induces a pleiotropic cellular response that is cell type-dependent. NDF and its receptor ErbB-4 are highly expressed in neurons, implying important roles in neuronal cell functions. In the present study we demonstrate that ErbB-4 receptors expressed in PC12 cells mediate NDF-induced signals and neurite outgrowth that are indistinguishable from those mediated by the nerve growth factor-activated Trk receptors. In PC12-ErbB-4 cells but not in PC12 cells, NDF induced an initial weak mitogenic signal and subsequently neurite outgrowth. The NDF-induced differentiation in PC12-ErbB-4 cells was mimicked by the pan-ErbB ligand betacellulin but not by other epidermal growth factor-like ligands. Thus, NDF and betacellulin mediate similar activities through the ErbB-4 receptor. Indeed, only these ligands induced strong phosphorylation of the ErbB-4 receptors. Neurite outgrowth induced by NDF in PC12-ErbB-4 cells was accompanied by sustained activation of mitogen-activated protein kinase (MAPK) and induction of the neural differentiation marker GAP-43. Inhibition of the MAPK kinase MEK or of protein kinase C (PKC) blocked NDF-induced differentiation, whereas elevation of cyclic AMP levels enhanced the response. Taken together, these results indicate that neurite outgrowth induced by ErbB-4 in PC12 cells requires MAPK and PKC signaling networks.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons. These authors contributed equally     

GTK, a Src-related tyrosine kinase, induces nerve growth factor-independent neurite outgrowth in PC12 cells through activation of the Rap1 pathway. Relationship to Shb tyrosine phosphorylation and elevated levels of focal adhesion kinase

The rat pheochromocytoma cell line PC12 is extensively used as a model for studies of neuronal cell differentiation. These cells develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor. The present study was performed in order to assess the role of mouse GTK (previously named BSK/IYK), a cytoplasmic tyrosine kinase belonging to the Src family, for neurite outgrowth in PC12 cells. We report that PC12 cells stably overexpressing GTK exhibit a larger fraction of cells with neurites as compared with control cells, and this response is not accompanied by an increased ERK activity. Treatment of the cells with the MEK inhibitor PD98059 did not reduce the GTK-dependent increased in neurite outgrowth. GTK expression induces a nerve growth factor-independent Rap1 activation, probably through altered CrkII signaling. We observe increased CrkII complex formation with p130(Cas), focal adhesion kinase (FAK), and Shb in PC12-GTK cells. The expression of GTK also correlates with a markedly increased content of FAK, phosphorylation of the adaptor protein Shb, and an association between these two proteins. Transient transfection of GTK-overexpressing cells with RalGDS-RBD or Rap1GAP, inhibitors of the Rap1 pathway, reduces the GTK-dependent neurite outgrowth. These data suggest that GTK participates in a signaling pathway, perhaps involving Shb, FAK and Rap1, that induces neurite outgrowth in PC12 cells.     

Ganglioside modulation of neural cell adhesion molecule and N-cadherin- dependent neurite outgrowth

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.     

Identification of the Cytoplasmic Regions of Fibroblast Growth Factor (FGF) Receptor 1 Which Play Important Roles in Induction of Neurite Outgrowth in PC12 Cells by FGF-1

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.     

Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line

The effects of extracellular Na+, K+ and Cl- on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na+ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60-150 mM Na+, whereas it decreased significantly at lower concentrations of Na+. Na+ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast neurite outgrowth was independent of K+ in the range 5-106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl- replaced by NO3-, SO2-4, or 2-hydroxyethanesulfonate. Thus, extracellular Na+ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca2+ concentration monitored by a fluorescent dye sensitive to Ca2+.     

Adapter protein SH2-B beta undergoes nucleocytoplasmic shuttling: implications for nerve growth factor induction of neuronal differentiation

The adapter protein SH2-B has been shown to bind to activated nerve growth factor (NGF) receptor TrkA and has been implicated in NGF-induced neuronal differentiation and the survival of sympathetic neurons. However, the mechanism by which SH2-B enhances and maintains neurite outgrowth is unclear. We examined the ability of truncation mutants to regulate neuronal differentiation and observed that certain truncation mutants localized in the nucleus rather than in the cytoplasm or at the plasma membrane as reported for wild-type SH2-B beta. Addition of the nuclear export inhibitor leptomycin B caused both overexpressed wild-type and endogenous SH2-B beta to accumulate in the nucleus of both PC12 cells and COS-7 cells as did deletion of a putative nuclear export sequence (amino acids 224 to 233) or mutation of two critical lysines in that sequence. Deleting or mutating the nuclear export signal caused SH2-B beta to lose its ability to enhance NGF-induced differentiation of PC12 cells. Neither the NGF-induced phosphorylation of ERKs 1 and 2 nor their subcellular distribution was altered in PC12 cells stably expressing the nuclear export-defective SH2-B beta(L231A, L233A). These data provide strong evidence that SH2-B beta shuttles constitutively between the nucleus and cytoplasm. However, SH2-B beta needs continuous access to the cytoplasm and/or plasma membrane to participate in NGF-induced neurite outgrowth. These data also suggest that the stimulatory effect of SH2-B beta on NGF-induced neurite outgrowth of PC12 cells is either downstream of ERKs or via some other pathway yet to be identified.     

PC12 cells utilize the homophilic binding site of L1 for cell-cell adhesion but L1-alphavbeta3 interaction for neurite outgrowth

Treatment of PC12 cells with nerve growth factor induces their differentiation into sympathetic neuron-like cells and the concomitant expression of the neural cell adhesion molecule L1, a member of the Ig superfamily. To investigate the mechanism of L1-stimulated neurite outgrowth in PC12 cells, substrate-immobilized fusion proteins containing different extracellular domains of L1 were assayed for their neuritogenic activity. Surprisingly, domain Ig2 of L1, which was previously found to contain both homophilic binding and neuritogenic activities, failed to promote neurite outgrowth. In contrast, L1-Ig6 stimulated neurite outgrowth from PC12 cells. Despite this, homotypic binding of PC12 cells was significantly inhibited by antibodies against L1-Ig2, indicating that L1-L1 binding contributed to the intercellular adhesiveness of PC12 cells, but L1-stimulated neurite outgrowth depends on heterophilic interactions. Thus, PC12 cells provide a valuable model for the study of these two distinct functions of L1. Mutagenesis of L1-Ig6 highlighted the importance of the Arg-Gly-Asp motif in this domain for neuritogenesis. Inhibition studies using cyclic Arg-Gly-Asp-containing peptide and anti-integrin antibodies suggested the involvement of alphavbeta3 integrin. Furthermore, neurite outgrowth stimulated by L1-Ig6 was inhibited by lavendustin A and the MEK inhibitor PD98059, suggesting a signaling pathway that involves tyrosine kinase activation and the mitogen-activated protein kinase cascade.     

甘薯提取物体外促进PC12和SK-N-SH细胞增殖及突起生长作用研究

观察甘薯提取物促进体外培养的PC12和SK-N-SH细胞的增值和突起生长作用.采用体外培养PC12和SK-N-SH细胞,以MTT法检测PC12和SK-N-SH细胞的活性率,以细胞突起长度超过2倍胞体或突起数目3个以上者为阳性细胞计算阳性细胞率来评价甘薯提取物对体外培养PC12和SK-N-SH细胞的增值和突起生长的促进作...     

KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner

The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3beta (GSK3beta) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3beta inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3beta overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3beta through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3beta and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3beta-dependent manner.