p75 and TrkA Receptor Signaling Independently Regulate Amyloid Precursor Protein mRNA Expression, Isoform Composition, and Protein Secretion in PC12 Cells

Abstract: The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75^NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75^NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75^NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75^NTR and TrkA receptors, binding of NGF to the p75^NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75^NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors—in contrast to p75^NTR-associated signaling—stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.     

Nerve Growth Factor-Induced Differentiation in Neuroblastoma Cells Expressing TrkA but Lacking p75NGFR

Abstract: Nerve growth factor (NGF) binds to two distinct cell surface receptors, TrkA, which is a receptor tyrosine kinase, and p75^NGFR, whose role in NGF-induced signal transduction remains unclear. We have found that human neuroblastoma IMR-32 cells express TrkA, but p75^NGFR expression was not detectable in these cells by northern blot analysis, immunoblotting, or chemical crosslinking experiments. Despite the lack of p75^NGFR expression, subnanomolar concentrations of recombinant human NGF induced neurite outgrowth, tyrosine phosphorylation, and immediate early gene expression in these cells. These results strongly suggest that NGF-induced neuronal differentiation in IMR-32 cells is initiated through TrkA in the absence of p75^NGFR. Thus, IMR-32 cells may provide a model for studying neurotrophic effects of NGF on adult striatal cholinergic neurons, which also lack p75^NGFR expression.     

Overexpression of wild-type PKD2 leads to increased proliferation and invasion of BON endocrine cells

Carcinoid tumors are rare neuroendocrine tumors with a predilection for the gastrointestinal tract. Protein kinase D (PKD), a novel serine/threonine protein kinase, has been implicated in the regulation of transport processes in certain cell types. We have reported an important role for PKD in stimulated peptide secretion from a human (BON) carcinoid cell line; however, the role of PKD isoforms, including PKD2, in the proliferation and invasion of carcinoid tumors remains unclear. In the present study, we found that overexpression of PKD2 by stable transfection of BON cells with PKD2-wild type (PKD2WT) significantly increased proliferation and invasion compared to cells transfected with PKD2-kinase dead (PKD2KD) or pcDNA3 (control). Similarly, inhibition of PKD2 activity with small interfering RNA (siRNA) significantly decreased proliferation and invasion compared to cells transfected with non-targeting control (NTC) siRNA. These data support an important role for PKD2 in carcinoid tumor progression. Targeted inhibition of the PKD family may prove to be a novel treatment option for patients with carcinoid tumors.     

Neurotrophic Factors Attenuate Glutamate-Induced Accumulation of Peroxides, Elevation of Intracellular Ca2+ Concentration, and Neurotoxicity and Increase Antioxidant Enzyme Activities in Hippocampal Neurons

Abstract: Exposure of cultured rat hippocampal neurons to glutamate resulted in accumulation of cellular peroxides (measured using the dye 2,7-dichlorofluorescein). Peroxide accumulation was prevented by an N -methyl- d -aspartate (NMDA) receptor antagonist and by removal of extracellular Ca²⁺, indicating the involvement of NMDA receptor-induced Ca²⁺ influx in peroxide accumulation. Glutamate-induced reactive oxygen species contributed to loss of Ca²⁺ homeostasis and excitotoxic injury because antioxidants (vitamin E, propyl gallate, and N-tert -butyl-α-phenylnitrone) suppressed glutamate-induced elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cell death. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor, each suppressed accumulation of peroxides induced by glutamate and protected neurons against excitotoxicity. bFGF, NGF, and BDNF each increased (to varying degrees) activity levels of superoxide dismutases and glutathione reductase. NGF increased catalase activity, and BDNF increased glutathione peroxidase activity. The ability of the neurotrophic factors to suppress glutamate toxicity and glutamate-induced peroxide accumulation was attenuated by the tyrosine kinase inhibitor genistein, indicating the requirement for tyrosine phosphorylation in the neuroprotective signal transduction mechanism. The data suggest that glutamate toxicity involves peroxide production, which contributes to loss of Ca²⁺ homeostasis, and that induction of antioxidant defense systems is a mechanism underlying the [Ca²⁺]_i-stabilizing and excitoprotective actions of neurotrophic factors.     

GM1 Ganglioside Activates the High-Affinity Nerve Growth Factor Receptor trkA

Abstract: The monosialoganglioside GM1 has been shown to possess neurotrophic activity in vitro and in vivo and is now used as an experimental treatment for a variety of neurological disorders and trauma. Little is known about the mechanism of action used by GM1. Because GM1 appears to enhance nerve growth factor (NGF) activity, we have used C6trk⁺ cells, a derivative of C6-2B glioma cells that express the high-affinity receptor for NGF trkA , to determine whether the neurotrophic effects of GM1 occurs through induction of trkA activity. Exposure of C6trk⁺ cells to NGF (10–50 ng/ml) resulted in a five- to 10-fold increase in trkA tyrosine phosphorylation within 5 min. Incubation of cells with GM1 resulted in a threefold increase in trkA phosphorylation beginning within 1 h and peaking between 3 and 6 h. Optimal responses to GM1 were obtained using 80–100 µ M concentrations. Moreover, tyrosine phosphorylation of known trkA target proteins, such as extracellular signal-regulated kinases, and suc -associated neurotrophic factor-induced tyrosine-phosphorylated target, were activated upon stimulation of C6trk⁺ cells with GM1. In addition, GM1 potentiated the NGF-mediated activation of tyrosine phosphorylation of trkA . GM1 failed to induce phosphorylation of trkA and target proteins in mock transfected cells. Thus, our data demonstrate that GM1 mimics some of the effects of NGF and suggest that the neurotrophic properties of GM1 may be attributed to its activation of trkA signal transduction.     

Multiple Levels for Regulation of TrkA in PC12 Cells by Nerve Growth Factor

Abstract: TrkA is a receptor tyrosine kinase for nerve growth factor (NGF). Recent studies indicate that NGF regulates not only activation of trkA kinase but also expression of the trkA gene. To further define NGF actions on trkA, we examined binding and signaling through trkA after both short and long intervals of NGF treatment. Induction of tyrosine phosphorylation on gp140 ^trkA was rapidly followed by down-regulation of cell surface and total cellular gp140 ^trkA . At later intervals, increased expression of trkA was evident in increased mRNA and protein levels. At 7 days, there was increased binding to gp140 ^trkA and increased signaling through this receptor. NGF appears to regulate trkA at several levels. In neurons persistently exposed to NGF, maintenance of NGF signaling may require increased trkA gene expression.     

Receptor-mediated autocrine growth-stimulatory effect of 5-hydroxytryptamine on cultured human pancreatic carcinoid cells.

5-hydroxytryptamine (5-HT) is a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5-HT. In this study, therefore, we examined the effect of 5-HT on growth of BON cells. Furthermore, by use of selective 5-HT receptor antagonists, we examined receptor and post-receptor mechanisms by which 5-HT-induced responses were produced. 5-HT stimulated growth of BON cells. 5-HT stimulated phosphatidylinositol (PI) hydrolysis in a dose-dependent fashion and inhibited cyclic AMP production in a dose-dependent fashion. The 5-HT1A/1B receptor antagonist, SDZ 21-009, prevented the reduction of cyclic AMP production evoked by 5-HT and inhibited the mitogenic action of 5-HT. The 5-HT1C/2 receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5-HT. The mitogenic action of 5-HT and the reduction of cyclic AMP production evoked by 5-HT were also inhibited by pertussis toxin. These results suggest that 5-HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5-HT involves receptor-mediated inhibition of the production of cyclic AMP which may be linked to pertussis toxin-sensitive GTP binding protein. 8-bromo-cyclic AMP inhibited growth of BON cells whereas 8-bromo-cyclic GMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a cAMP-dependent protein kinase antagonist (Rp-cAMPS) could stimulate BON cell growth.     

Nerve Growth Factor Protects PC12 Cells Against Peroxynitrite-Induced Apoptosis via a Mechanism Dependent on Phosphatidylinositol 3-Kinase

Abstract: Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as PI 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3-kinase activity was reduced by 50% of that detected in control cells, whereas PI 3-kinase activity in NGF-treated cells was unaffected by peroxynitrite. If an antibody against PI 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21^Ras-dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21^Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21^Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.     

K-252b Potentiation of Neurotrophin-3 Is trkA Specific in Cells Lacking p75NTR

Abstract: K-252b potentiates the neurotrophic effects of neurotrophin-3 (NT-3) in primary cultures of rat central cholinergic and peripheral sensory neurons and in a rat pheochromocytoma PC12 cell line. The ligand and receptor specificity, and role of the low-affinity neurotrophin receptor (p75^NTR) in the potentiation response induced by K-252b, are unknown. To address the issues of ligand and receptor specificity of K-252b potentiation, we have examined neurotrophin-induced DNA synthesis ([³H]thymidine incorporation) in NIH3T3 cells expressing trkA, trkB, or trkC . Neither NT-3 nor K-252b alone could stimulate mitogenic activity in the trkA -overexpressing clone. However, coaddition of K-252b (EC₅₀ of ∼2 n M ) with 10–100 ng/ml NT-3 led to incorporation of [³H]thymidine in trkA expressing cells to a level induced by optimal concentrations of nerve growth factor (NGF). The K-252b- and NT-3-induced [³H]thymidine incorporation correlated with an increase in the tyrosine autophosphorylation of the trkA receptor as well as tyrosine phosphorylation of trk -associated phospholipase C-γ1 and SH2-containing proteins. K-252b did not potentiate submaximal doses of NGF, or maximal doses of brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5) in trkA -expressing cells. Furthermore, K-252b did not potentiate DNA synthesis by submaximal doses of BDNF, NT-4/5, or NT-3 in trkB - or trkC -expressing NIH3T3 cells, suggesting that the potentiation profile for K-252b was specific for NT-3 in trkA -expressing cells. We found no expression of p75^NTR in the trk -expressing NIH3T3 cells. This is the first demonstration that K-252b potentiates a trkA -mediated biological nonneuronal response by NT-3 that occurs independent of p75^NTR and appears to be both ligand and receptor specific.     

Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP-Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells

Abstract: To study how growth factors affect stimulus-secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP-induced [Ca²⁺]_i rise and dopamine secretion in PC12 cells. After a 4-day incubation of cells, all three factors increased ATP-induced dopamine secretion significantly. We then examined which step of ATP-induced secretion was affected by the growth factors. Cellular levels of dopamine-β-hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP-induced [Ca²⁺]_i rise was also enhanced after growth factor treatment. The EC₅₀ of ATP for inducing [Ca²⁺]_i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca²⁺]_i of dopamine secretion was increased significantly only in NGF-treated cells. Our results suggest that for EGF- and insulin-treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca²⁺]_i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca²⁺ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca²⁺]_i.     

Induction of a Nerve Growth Factor-Sensitive Kinase that Phosphorylates the DNA-Binding Domain of the Orphan Nuclear Receptor NGFI-B

Abstract: Nerve growth factor (NGF) induces the synthesis and the phosphorylation of the orphan nuclear receptor NGFI-B in PC12 cells. Previous work has shown that phosphorylation, by protein kinase A, of a specific serine in the DNA-binding domain inhibits its binding to the NGFI-B response element. Also, cytoplasmic extracts from PC12 cells phosphorylate this serine, and phosphorylation is greater in extracts from cells treated with NGF. The present work describes the induction, identification, and partial purification of a kinase (termed NGFI-B kinase I) from PC12 cell extracts that catalyzes this phosphorylation. Phosphorylation of the DNA-binding domain with this purified preparation inhibits its binding to the NGFI-B response element. The kinase is rapidly activated by treatment of the cells with NGF, and the activation lasts for at least several hours. It also is activated by fibroblast growth factor and epidermal growth factor (EGF), but the activation by EGF is quite transient. The kinase requires Mg²⁺ but will use Mn²⁺. The molecular mass of the kinase is 95–100 kDa, and it is different from protein kinase A, Fos kinase, or pp90 ^rsk . Comparison with a partially purified preparation of cyclic AMP response element-binding protein kinase, however, indicates that the two are either very similar or identical.     

Down-Regulation of c-neu Receptors by Nerve Growth Factor in PC12 Cells

Abstract: A small number of p185^{c- neu} receptors have been found on PC12 cells. These receptors show some basal phosphorylation in quiescent cells. When the cells are treated with nerve growth factor (NGF) for a short time, some increase in phosphorylation is seen, mainly on serine and threonine residues, and this is accompanied by a slight shift in the apparent molecular weight. Epidermal growth factor (EGF) also increases the phosphorylation of p185^{c- neu} , in this case on tyrosine residues. Neither heregulin-β1 nor gp30 stimulates the tyrosine phosphorylation of p185^{c- neu} , and neither has a proliferative effect on the cells. Treatment of the cells with NGF for 5 days produces a 70–80% reduction in the number of p185^{c- neu} receptors. This down-regulation does not occur when PC12nnr5 cells, which lack the high-affinity NGF receptor, p140 ^trk , are treated with NGF.The level of p185^{c- neu} mRNA is not altered by NGF treatment, suggesting that the down-regulation is due to either a translational or a posttranslational alteration.     

Transduction Mechanisms Involved in Thrombin Receptor-Induced Nerve Growth Factor Secretion and Cell Division in Primary Cultures of Astrocytes

Abstract: In astrocytes, thrombin and thrombin receptor-activating activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [³H]thymidine incorporation into DNA (EC₅₀ = 1 n M and +650% at 100 n M for thrombin; EC₅₀ = 3 µ M and +600% at 100 µ M for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 n M thrombin or 100 µ M TRAP-14). The [³H]thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as G_i or G_o, is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.     

Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar

Abstract. Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of nons-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75^NGFR and p140^prototrk) on responsive cells. The continously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF. proNGF and p75^NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75^NGFR was correlated with differentiation events only in dental mesenchyme: polarizing odontoblasts expressed p75^NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75^NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75^NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.     

Phorbol ester-mediated neurotensin secretion is dependent on the PKC-alpha and -delta isoforms

Neurotensin (NT) plays an important role in gastrointestinal secretion, motility, and growth. The mechanisms regulating NT secretion are not entirely known. Our purpose was to define the role of the PKC signaling pathway in secretion of NT from BON cells, a human pancreatic carcinoid cell line that produces and secretes NT peptide. We demonstrated expression of all 11 PKC isoforms at varying levels in untreated BON cells. Expression of PKC-alpha, -beta2, -delta, and -mu isoforms was most pronounced. Immunofluorescent staining showed PKC-alpha and -mu expression throughout the cytoplasm and in the membrane. Also, significant fluorescence of PKC-delta was noted in the nucleus and cytoplasm. Treatment with PMA induced translocation of PKC-alpha, -delta, and -mu from cytosol to membrane. Activation of PKC-alpha, -delta, and -mu was further confirmed by kinase assays. Addition of PKC-alpha inhibitor G?-6976 at a nanomolar concentration, other PKC inhibitors G?-6983 and GF-109203X, or PKC-delta-specific inhibitor rottlerin significantly inhibited PMA-mediated NT release. Overexpression of either PKC-alpha or -delta increased PMA-mediated NT secretion compared with control cells. We demonstrated that PMA-mediated NT secretion in BON cells is associated with translocation and activation of PKC-alpha, -delta, and -mu. Furthermore, inhibition of PKC-alpha and -delta blocked PMA-stimulated NT secretion, suggesting a critical role for these isoforms in NT release.     

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

Monokine induced by interferon-gamma acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons

We found that a monokine induced by interferon-gamma (Mig, CXCL9), which belongs to the CXC chemokine subfamily, acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons. PC12 cells were shown to express a single class of high affinity binding sites for Mig (670 receptors/cell, Kd = 2.9 nm). Mig induced neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of extracellular signal-regulated kinase signaling pathways between Mig and nerve growth factor (NGF) revealed that these pathways are crucial for Mig action as well as NGF. K252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks) did not inhibit the action of Mig, suggesting that Mig action occurs via a different receptor from that of NGF. Furthermore, Mig as well as NGF promoted PC12 survival under serum-free conditions and activated Akt/protein kinase B downstream from phosphatidylinositol 3-kinase (PI3K). Because the PI3K inhibitor LY294002 prevented the Mig- and NGF-induced survival effect, this effect is probably mediated by the PI3K signaling pathway. Mig also promoted survival of rat primary sympathetic neurons that die when deprived of NGF. These results suggest that chemokines, including Mig (CXCL9) have neurotrophic effects on the nervous system.     

Nestin-expressing neural stem cells identified in the scar following myocardial infarction

Nerve fiber innervation of the scar following myocardial damage may have occurred either via the growth of pre-existing fibers and/or the mobilization of neural stem cells. The present study examined whether neural stem cells were recruited to the infarct region of the rat heart following coronary artery ligation. The neural stem cell marker nestin was detected in the infarct region of 1-week post-myocardial infarct (MI) male rats and cultured scar-derived neural-like cells. By contrast, nestin staining was undetected in either scar myofibroblasts or cardiac myocytes residing in the non-infarcted left ventricle. Reactive astrocytes were isolated from the infarct region and characterized by the co-expression of nestin, glial fibrillary acidic protein, and vimentin. Specific staining of oligodendrocytes and neurons was also detected in the infarct region and cultured scar-derived neural-like cells. Furthermore, neurofilament-M positive fibers were identified in the scar and tyrosine hydroxylase immunoreactivity was observed in peripherin-positive neurons. Neurite formation was induced in PC12 cells treated with the conditioned-media of primary passage scar-derived cells, highlighting the synthesis and secretion of neurotrophic factors. Nerve growth factor (NGF) and brain-derived neurotrophic factor were detected in myofibroblasts and neural cells, and both cell types expressed the NGF receptors trkA and p75. These data highlight the novel observation that neural stem cells were recruited to the infarct region of the damaged rat heart and may contribute in part to nerve fiber growth and subsequent innervation of the scar.