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.     

Laminin-1 and the RKRLQVQLSIRT Laminin-1 α1 Globular Domain Peptide Stimulate Matrix Metalloproteinase Secretion by PC12 Cells

Here we have investigated the ability of laminin-1 and specific laminin-1-derived synthetic peptides to stimulate neuronal cell matrix metalloproteinase secretion. Zymographic analysis of conditioned media from laminin-1-treated PC12 and NG108-15 cells revealed a 72-kDa matrix metalloproteinase which was not secreted by untreated cells. Laminin-1 α1 chain-derived synthetic peptides, AASIKVAVSADR (LAM-L) and RKRLQVQLSIRT (AG-73), also stimulated PC12 cell secretion of a 72-kDa matrix metalloproteinase. We further investigated the structural requirements of AG-73 for cell attachment, neurite outgrowth, and matrix metalloproteinase secretion using a series of AG-73 analogs that had single amino acids substituted with alanine. At the substrate levels tested, the AG-73 peptide promoted the adhesion of 67% of the PC12 cells and neurite outgrowth in 71% of the PC12 cells. Substitutions in any one of the amino acids within the central LQVQ sequence resulted in a large reduction in cell attachment whereas substitution in the carboxyl terminal proximal amino acids L, S, and R had little effect on attachment. Alanine substitution of any of the amino terminal proximal LQV amino acids and the carboxyl terminal L, I, and R residues resulted in a 65–91% reduction in neurite outgrowth. These data demonstrate that the sequence requirements for cell attachment and neurite outgrowth were not necessarily coupled but that the sequence requirements for neurite outgrowth and matrix metalloproteinase secretion were identical. We conclude that laminin-1 is able to stimulate neuronal cells to secrete a matrix metalloproteinase. Further, this study identifies the LQVXLXIR laminin-1 α1 globular domain peptide to be capable of stimulating both neurite outgrowth and matrix metalloproteinase secretion.     

Iron enhances NGF-induced neurite outgrowth in PC12 cells

Rat pheochromocytoma 12 (PC12) cells undergo neuronal differentiation in response to nerve growth factor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular signal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting effect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF-induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.     

Ubiquitin and ubiquitin-protein conjugates in PC12h cells: Changes during neuronal differentiation

Ubiquitin and ubiquitin-protein conjugates in PC12h cells were detected with in vitro [¹²⁵I]ubiquitination, and quantified by immunoblotting. These levels were altered by nerve growth factor (NGF), which promotes neuronal differentiation. (i) Levels of high molecular weight (HMW) ubiquitin-protein conjugates ranging from 40 to 1,000kDa were increased by 2 days of NGF treatment, and remained high up to 10 days of NGF treatment. (ii) Ubiquitin and a 23-kDa conjugate tended to be decreased from days 2 to 10 of NGF treatment. 10-Day culture with 10 nM staurosporine, an protein kinase inhibitor, that blocks NGF-induced neurite outgrowth suppressed the NGF-induced increases in levels of HMW conjugates. Cyclic AMP and forskolin, both of which promote neurite outgrowth, mimicked the NGF-induced changes in ubiquitin and HMW conjugates, but phorbol ester and epidermal growth factor had little effect. These findings suggest that changes in ubiquitin-protein conjugates are closely coupled with neuronal differentiation.     

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.     

Effects of fluvoxamine on nerve growth factor-induced neurite outgrowth inhibition by dexamethasone in PC12 cells

In the present study, we examined the effects of fluvoxamine on nerve growth factor (NGF)-induced neurite outgrowth inhibition by dexamethasone (DEX) in PC12 cells. Fluvoxamine increased NGF-induced neurite outgrowth. Compared with co-treatment with NGF and fluvoxamine, p-Akt levels were higher than the values without fluvoxamine. The phosphorylated extracellular regulated kinase 1/2 levels were slightly increased by co-treatment with NGF and fluvoxamine. Fluvoxamine concentration-dependently improved NGF-induced neurite outgrowth inhibition by DEX. Fluvoxamine also improved the decrease in the NGF-induced p-Akt level caused by DEX. Interestingly, the sigma-1 receptor antagonist NE-100 blocked the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX. The selective sigma-1 receptor agonist PRE-084 also improved NGF-induced neurite outgrowth inhibition by DEX, which is blocked by NE-100. These results indicate that the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX may be attributable to the phosphorylation of Akt and the sigma-1 receptor.     

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.     

Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.     

Reconstituted basement membrane enhances neurite outgrowth in PC12 cells induced by nerve growth factor

Rat pheochromocytoma cells (PC12) respond to nerve growth factor (NGF) by elaborating neurites. We have studied the effect of extracellular matrix proteins on responsiveness of PC12 cells to NGF. NGF elicited neurite outgrowth in cells plated on collagen, fibronectin, or laminin within 48-72 h. In contrast, cells entrapped in reconstituted basement membrane prepared from the Engelbreth-Holm-Swarm tumor (EHS-BM) responded within 24 h. Cells plated on collagen or fibronectin required a minimal dose of 50 ng/ml NGF to achieve the same effect as cells entrapped in EHS-BM at 1 ng/ml NGF. Neurites displayed by cells plated in EHS-BM were more extensively branched and remained intact up to 21 days following a single administration of NGF.     

Tumor promoter modulation of epidermal growth factor- and nerve growth factor-induced adhesion and growth factor binding of PC-12 pheochromocytoma cells

Nerve growth factor (NGF) has previously been shown to increase the rate of adhesion of PC-12 pheochromocytoma cells to cell culture dishes. This increase in the rate of adhesion was postulated to be important in NGF-mediated neurite outgrowth. We now report that epidermal growth factor (EGF) is also able to increase the rate of adhesion of PC-12 cells to cell culture dishes, but does not elicit neurite outgrowth. The dose-response curve for EGF is bell-shaped, in contrast to the more classically shaped dose-response curve obtained with NGF. Tetradecanoyl-phorbol-acetate (TPA), a potent tumor promoter, blocks the EGF-induced increase in adhesion rate of PC-12 cells, but does not alter the NGF-induced increase in adhesion rate. TPA shifts the EGF binding curve to the right for PC-12 cells, but does not alter maximal EGF binding at saturating concentrations of EGF. The binding of NGF to PC-12 cells is not affected by TPA. NGF-induced neurite formation by PC-12 cells is unaffected by TPA, in contrast to the previously reported delay of neurite outgrowth of serum-deprived neuroblastoma cells and NGF-exposed chick embryonic ganglia cells. NGF and EGF both cause a decrease in the number of short microvilli and an increase in the number of long microvilli on PC-12 cells. TPA blocks the decrease in the number of short microvilli in EGF-treated cells, but not in NGF-treated cells. Long microvilli formation is blocked by TPA in both conditions, suggesting the latter are not involved in the increased adhesion rates.     

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.     

RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway

The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.     

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.     

Simultaneous suppression of cdc2 and cdk2 activities induces neuronal differentiation of PC12 cells

The involvement of cdc2 and cdk2 during neuronal differentiation in rat pheochromocytoma PC12 cells was examined. When PC12 cells were cultured with nerve growth factor (NGF), expression of cdc2 decreased significantly after day 5, while expression of cdk2 decreased gradually after day 7. Cells overexpressing cdc2 or cdk2 were resistant to NGF-induced differentiation and growth suppression, and maintained high cdc2 or cdk2 kinase activity, respectively, during NGF treatment. In contrast, the NGF-treated parental cells showed a marked decline in these kinase activities after day 3. When PC12 cells were treated with specific inhibitors of cdc2/cdk2 (butyrolactone-I, olomoucin), they showed marked neurite extension and up-regulation of microtubule-associated protein 2 expression. In addition, treatment with mixtures of antisense oligonucleotides for cdc2 and cdk2 resulted in down-regulation of both cdc2 and cdk2 kinase activities as well as significant neurite outgrowth and up-regulation of microtubule-associated protein 2 expression. However, neurite outgrowth was not observed in cells treated with either single antisense oligonucleotide, or antisense cdc2 + cdk4 or cdk2 + cdk4 oligonucleotide mixtures. These results suggest that simultaneous down-regulation of cdc2 and cdk2 activity is sufficient and necessary for neuronal differentiation in PC12 cells.     

Synergistic effect of immobilized laminin and nerve growth factor on PC12 neurite outgrowth

Immobilized extracellular matrix proteins and neurotrophins have been extensively studied to enhance neuronal adhesion and proliferation on surfaces for applications in nerve tissue engineering and neuroprosthetic devices. This article describes how the coimmobilization of laminin, an extracellular matrix protein and nerve growth factor (NGF), a neurotrophin can enhance neurite outgrowth observed separately with each type of molecule. In the absence of immobilized NGF, PC12 neurite outgrowth is influenced strongly by the presence of NGF in solution and unaffected by significant increases in laminin surface density (18.7–93.5 ng/mm²). However, when both laminin and NGF are immobilized together, the surface density of laminin is an important factor in determining whether or not the neurite outgrowth‐promoting effect of NGF can be obtained. PC12 neurite outgrowth on surfaces with coimmobilized laminin and NGF with surface densities of 27.6 ng/mm² and 1.4 ng/mm², respectively, are similar to that observed on surfaces with immobilized laminin and dissolved NGF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation.     

Essential role of NKCC1 in NGF-induced neurite outgrowth

The Na(+)/K(+)/2Cl(-) cotransporter (NKCC) mediates electroneutral transport of 2Cl(-) coupled with Na(+) and K(+) across the plasma membrane, and plays crucial roles in Cl(-) uptake into the cells, homeostasis of cellular Cl(-), and cell volume regulation. However, we have very limited information on the roles of ion transporters in neurite outgrowth in neuronal cells. In the present study, we report the role of NKCC1 (an isoform of NKCC) in NGF-induced neurite outgrowth of rat pheochromocytoma PC12D cells. The expression level of NKCC1 protein was increased by NGF treatment. Knock-down of NKCC1 by RNA interference (RNAi) drastically diminished the NGF-induced neurite outgrowth. Transfection of enhanced green fluorescent protein (EGFP)-tagged rat NKCC1 into cells for clarification of intracellular localization of NKCC1 revealed that the EGFP-rNKCC1 was mainly localized in the plasma membrane at growth cone during neurite outgrowth. These observations suggest that NKCC1 plays a fundamental role in NGF-induced neurite outgrowth of PC12D cells.