Nerve Growth Factor-stimulated PC12 Cells Research Articles

Neuroprotective effect of Na+ /H+ exchangers isoform-1 inactivation against 6-hydroxydopamine-induced mitochondrial dysfunction and neuronal apoptosis in Parkinson's disease models.

Parkinson's disease (PD) is a disabling neurodegenerative disease mainly caused by degeneration of mesencephalic dopaminergic neurons in the substantia nigra pars compacta (SNpc). The neuroprotective role of Na+ /H+ exchangers isoform-1 (NHE1) inactivation in cerebral ischemic damage has been elucidated. The current study aimed to investigate the impacts of NHE1 in PD. In this study, 6-hydroxydopamine (6-OHDA)-induced PD rat models were established to attempt to illuminate the role and underlying mechanisms of NHE1 in SNpc neurons of PD. Meanwhile, nerve growth factor-stimulated PC12 cells followed by 6-OHDA treatment was used to mimic PD in vitro. Results showed that the protein levels of NHE1 were significantly increased in the SNpc neurons of rats and differentiated PC12 cells after 6-OHDA treatment. Inactivation of NHE1 with chemical inhibitor HOE642 suppressed SNpc neuronal loss and NHE1 expression in PD rats. The overlays of tyrosine hydroxylase and NHE1 displayed that NHE1 expression was not colocalized but closely associated with TH. Besides, treatment with HOE642 relieved the dyskinesia, mitochondrial dysfunction, and neuronal apoptosis. Further in vitro evidence confirmed that inhibition of NHE1 by genetic-knockdown prevented mitochondrial dysfunction and apoptosis. Our study represents the first experimental evidence of a potential role for NHE1 in the pathogenesis of PD.

Targeting MicroRNA-125b Promotes Neurite Outgrowth but Represses Cell Apoptosis and Inflammation via Blocking PTGS2 and CDK5 in a FOXQ1-Dependent Way in Alzheimer Disease.

This study aimed to explore the molecular regulatory network among microRNA-125b (miR-125b), forkhead box Q1 (FOXQ1), prostaglandin-endoperoxide synthase 2 (PTGS2), and cyclin-dependent kinase 5 (CDK5), as well as their effects on cell apoptosis, neurite outgrowth, and inflammation in Alzheimer disease (AD). Rat embryo cerebral cortex neurons and nerve growth factor–stimulated PC12 cells were insulted by Aβ1−42 to construct two AD cellular models. Negative control (NC) inhibitor, miR-125b inhibitor, NC siRNA, FOXQ1 siRNA, PTGS2 siRNA, and CDK5 siRNA were transferred into the two AD cellular models alone or combined. Then, cell apoptosis, neurite outgrowth, proinflammatory cytokines, miR-125b, FOXQ1, PTGS2, and CDK5 expressions were detected. MiR-125b inhibition facilitated neurite outgrowth but suppressed cell apoptosis and proinflammatory cytokines (tumor necrosis factor-α, interleukin 1β, and interleukin 6); meanwhile, it upregulated FOXQ1 but downregulated PTGS2 and CDK5. Furthermore, FOXQ1 inhibition promoted cell apoptosis and proinflammatory cytokines but repressed neurite outgrowth; PTGS2 inhibition achieved the opposite effects; CDK5 inhibition attenuated cell apoptosis, whereas it less affected neurite outgrowth and inflammation. Notably, FOXQ1 inhibition attenuated, whereas PTGS2 inhibition elevated the effect of miR-125b inhibition on regulating neurite outgrowth, cell apoptosis, and proinflammatory cytokines. As for CDK5 inhibition, it enhanced the effect of miR-125b inhibition on regulating cell apoptosis, but less impacted the neurite outgrowth and proinflammatory cytokines. Additionally, PTGS2 inhibition and CDK5 inhibition both reversed the effect of FOXQ1 inhibition on regulating cell apoptosis, neurite outgrowth, and proinflammatory cytokines. In conclusion, targeting miR-125b alleviates AD progression via blocking PTGS2 and CDK5 in a FOXQ1-dependent way.

Rab20, a novel Rab small GTPase that negatively regulates neurite outgrowth of PC12 cells.

The Rab family small GTPases are key players in the membrane traffic that underlies various cellular phenomena. Neurite outgrowth, which is a prerequisite for neuronal network formation, also requires membrane traffic from the cell body to the tips of neurites. Although several Rabs have been shown to promote neurite outgrowth, very little is known about Rab involvement in the negative regulation of neurite outgrowth. Here we used nerve growth factor-stimulated PC12 cells to perform siRNA-based comprehensive knockdown screenings for Rabs that negatively regulate neurite outgrowth and succeeded in identifying Rab20 as a novel negative regulator of neurite outgrowth. Our findings showed that knockdown of endogenous Rab20 in PC12 cells promoted neurite outgrowth, whereas overexpression of active Rab20 inhibited it. We also found that the presence of Gly-64 and Cys-70, both of which are conserved only in the switch II region, a putative effector binding domain, of Rab20 is required for the inhibitory effect of Rab20 on neurite outgrowth. These findings indicated that active Rab20 suppresses neurite outgrowth of PC12 cells, possibly through interaction with an unidentified effector molecule that specifically recognizes certain amino acids in the switch II region of Rab20.

Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

BackgroundExposure to chemicals might be toxic to the developing brain. There is a need for simple and robust in vitro cellular models for evaluation of chemical-induced neurotoxicity as a complement to traditional studies on animals. In this study, neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) were compared with human neuroblastoma SH-SY5Y cells and rat adrenal pheochromocytoma PC12 cells for their ability to detect toxicity of methylmercury (MeHg), okadaic acid and acrylamide.MethodsRetinoic acid-treated P19 and SH-SY5Y cells and nerve growth factor-stimulated PC12 cells, allowed to differentiate for 6 days, were exposed to MeHg, okadaic acid and acrylamide for 48 h. Cell survival and neurite outgrowth were assessed with the calcein-AM assay and fluorescence detection of antibodies against the cytoskeletal neuron-specific protein βIII-tubulin, respectively. The effects of glutathione (GSH) and the potent inhibitor of GSH synthesis buthionine sulfoximine (BSO) on the MeHg induced-toxicity were assessed using the PrestoBlue™ cell viability assay and the TMRE mitochondrial membrane potential assay.ResultsDifferentiated P19 cells developed the most extensive neuronal network among the three cell models and were the most sensitive neuronal model to detect neurotoxic effects of the test compounds. MeHg produced a concentration-dependent toxicity in differentiated P19 cells and SH-SY5Y cells, with statistically significant effects at concentrations from 0.1 μM in the P19 neurons and 1 μM in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (ΔΨm) in the differentiated P19 cells and SH-SY5Y cells, that were attenuated by GSH. Okadaic acid and acrylamide also showed statistically significant toxicity in the P19 neurons, but not in the SH-SY5Y cells or the P12 cells.ConclusionsP19 neurons are more sensitive to detect cytotoxicity of MeHg, okadaic acid and acrylamide than retinoic acid-differentiated SH-SY5Y cells and nerve growth factor-treated PC12 cells. P19 neurons are at least as sensitive as differentiated SH-SY5Y cells to detect the loss of mitochondrial membrane potential produced by MeHg and the protective effects of extracellular GSH on MeHg toxicity. P19 neurons may be a useful model to study neurotoxic effects of chemicals.

Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells

The small GTPase Rab35 is a molecular switch for membrane trafficking that regulates a variety of cellular events. We previously showed that Rab35 promotes neurite outgrowth of nerve growth factor-stimulated PC12 cells through interaction with centaurin-β2 (also called ACAP2). Centaurin-β2 is the only Rab35-binding protein reported thus far that exclusively recognizes Rab35 and does not recognize any of the other 59 Rabs identified in mammals, but the molecular basis for the exclusive specificity of centaurin-β2 for Rab35 has remained completely unknown. In this study, we performed deletion and mutation analyses and succeeded in identifying the residues of Rab35 and centaurin-β2 that are crucial for formation of a Rab35·centaurin-β2 complex. We found that two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are responsible for binding centaurin-β2 and that the same residues are dispensable for Rab35 recognition by other Rab35-binding proteins. We also determined the minimal Rab35-binding site of centaurin-β2 and identified two asparagine residues (Asn-610 and Asn-691) in the Rab35-binding site as key residues for its specific Rab35 recognition. We further showed by knockdown-rescue approaches that neither a centaurin-β2 binding-deficient Rab35(T76S/T81A) mutant nor a Rab35 binding-deficient centaurin-β2(N610A/N691A) mutant supported neurite outgrowth of PC12 cells, thereby demonstrating the functional significance of the Rab35/centaurin-β2 interaction during neurite outgrowth of PC12 cells.

Isolation, structural characterization and neurotrophic activity of a polysaccharide from Phellinus ribis

A new polysaccharide named PRG was isolated from the fruiting bodies of Phellinus ribis by hot water extraction, ethanol precipitation, anion-exchange and gel-filtration chromatography. PRG was homogeneous, with a molecular weight of 5.16×103Da, as determined by high-performance size-exclusion chromatography–multiangle laser light scattering analysis. Its structural characteristics were investigated and elucidated by methylation analysis, partial acid hydrolysis, gas–liquid chromatography mass spectrometry, Fourier transform infrared and nuclear magnetic resonance spectroscopy. Based on obtained data, PRG was found to be a β-d-glucan containing a (1→3)-linked backbone, with a branch of two (1→6)-linked and one terminal glucoses substituting at the C-6 position every three residues, along the main chain. PRG exhibited neurotrophic activity, which significantly promoted the neurite outgrowth of the nerve growth factor-stimulated PC12 cells, suggesting that it might be a potential candidate for the treatment of neurodegenerative diseases.

Antibodies Directed to Neisseria gonorrhoeae Impair Nerve Growth Factor-Dependent Neurite Outgrowth in Rat PC12 Cells

In children born from mothers with prenatal infections with the Gram-negative bacterium Neisseria gonorrhoeae, schizophrenia risk is increased in later life. Since cortical neuropil formation is frequently impaired during this disease, actions of a rabbit polyclonal antiserum directed to N. gonorrhoeae on neurite outgrowth in nerve growth factor-stimulated PC12 cells were investigated here. It turned out that 10μg/ml of the antiserum leads indeed to a significant reduction in neurite outgrowth, whereas an antiserum directed to Neisseria meningitidis had no such effect. Furthermore, reduction in neurite outgrowth could be reversed by the neuroleptic drugs haloperidol, clozapine, risperidone, and olanzapine. On the molecular level, the observed effects seem to include the known neuritogenic transcription factors FoxO3a and Stat3, since reduced neurite outgrowth caused by the antiserum was accompanied by a reduced phosphorylation of both factors. In contrast, restitution of neurite outgrowth by neuroleptic drugs revealed no correlation to the phosphorylation state of these factors. The present report gives a first hint that bacterial infections could indeed lead to impaired neuropil formation in vitro; however, the in vivo relevance of this finding for schizophrenia pathogenesis remains to be clarified in the future.

Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells

BackgroundThe major goal of the study was to compare the genetic programs utilized by the neuropoietic cytokine Interleukin-6 (IL-6) and the neurotrophin (NT) Nerve Growth Factor (NGF) for neuronal differentiation.ResultsThe designer cytokine Hyper-IL-6 in which IL-6 is covalently linked to its soluble receptor s-IL-6R as well as NGF were used to stimulate PC12 cells for 24 hours. Changes in gene expression levels were monitored using Affymetrix GeneChip technology. We found different expression for 130 genes in IL-6- and 102 genes in NGF-treated PC12 cells as compared to unstimulated controls. The gene set shared by both stimuli comprises only 16 genes.A key step is upregulation of growth factors and functionally related external molecules known to play important roles in neuronal differentiation. In particular, IL-6 enhances gene expression of regenerating islet-derived 3 alpha (REG3A; 1084-fold), regenerating islet-derived 3 beta (REG3B/PAPI; 672-fold), growth differentiation factor 15 (GDF15; 80-fold), platelet-derived growth factor alpha (PDGFA; 69-fold), growth hormone releasing hormone (GHRH; 30-fold), adenylate cyclase activating polypeptide (PACAP; 20-fold) and hepatocyte growth factor (HGF; 5-fold). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). Both stimuli activate growth-associated protein 43 (GAP-43) indicating that PC12 cells undergo substantial neuronal differentiation.Moreover, IL-6 activates the transcription factors retinoic acid receptor alpha (RARA; 20-fold) and early growth response 1 (Egr1/Zif268; 3-fold) known to play key roles in neuronal differentiation.Ingenuity biological function analysis revealed that completely different repertoires of molecules are recruited to exert the same biological functions in neuronal differentiation. Major sub-categories include cellular growth and differentiation, cell migration, chemotaxis, cell adhesion, small molecule biochemistry aiming at changing intracellular concentrations of second messengers such as Ca2+ and cAMP as well as expression of enzymes involved in posttranslational modification of proteins.ConclusionThe current data provide novel candidate genes involved in neuronal differentiation, notably for the neuropoietic cytokine IL-6. Our findings may also have impact on the clinical treatment of peripheral nerve injury. Local application of a designer cytokine such as H-IL-6 with drastically enhanced bioactivity in combination with NTs may generate a potent reparative microenvironment.

Neurofibromatosis Type 1 (NF1) Tumor Suppressor, Neurofibromin, Regulates the Neuronal Differentiation of PC12 Cells via Its Associating Protein, CRMP-2

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.

A Novel Candidate Compound with Urethane Structure for Anticancer Drug Development

Diethyl-4,4'-methylenebis(N-phenylcarbamate) (MDU) is a urethane compound that we originally synthesized, along with three other compounds, to investigate how polyurethane is hydrolysed. We tested the four compounds for cytotoxicity in two Chinese hamster cell lines (CHL and V79) and a human cancer cell line (HeLa S3). MDU showed the strongest cytotoxicity in all the cell lines with an IC50 of around 0.1 microg/ml. We further investigated MDU for its ability to induce chromosome aberrations (CAs) and micronuclei (MN) in CHL cells. MDU induced around 100% polyploid cells at 0.5 microg/ml after 24- and 48-h treatment in the CA test and a significantly increased frequency of micronuclei, polynuclear cells, and mitotic cells in the MN test, suggesting that it may induce numerical CAs. MDU's ability to cause mitotic arrest in CHL cells was greater than that of taxol and colchicine. Based on a COMPARE analysis using JFCR39, a panel of cancer cell lines, we predicted MDU to be a tubulin inhibitor. We confirmed this possibility in nerve growth factor-stimulated PC12 cells as well as in HT1080 cells, in which MDU exhibited the activity to inhibit tubulin polymerization. MDU is simpler in structure than existing anticancer drugs taxol and vincristine and can be synthesized relatively easily. Here we offer MDU as a potential new type of anticancer drug, stable even at room temperature, and inexpensive.

An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor–stimulated PC12 cells

The local accumulation of phosphatidylinositol (3,4,5) trisphosphate (PIP3) and resulting activation of Rac1/Cdc42 play a critical role in nerve growth factor (NGF)–induced neurite outgrowth. To further explore the mechanism, we visualized PIP3, phosphatidylinositol (3,4) bisphosphate, and Rac1/Cdc42 activities by fluorescence resonance energy transfer (FRET) imaging in NGF-stimulated PC12 cells. Based on the obtained FRET images, and with the help of in silico kinetic reaction model, we predicted that PI-5-phosphatase negatively regulates PIP3 upon NGF stimulation. In agreement with this model, depletion of Src homology 2 domain–containing inositol polyphosphate 5-phosphatase 2 (SHIP2) markedly potentiated NGF-induced Rac1/Cdc42 activation and PIP3 accumulation and considerably increased the number and the length of neurites in phosphate and tensin homologue–depleted PC12 cells. Further refinement of the computational model predicted Rac1 regulation of PI3-kinase and SHIP2, which was also validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP3 coordinately works with the PI3-kinase–mediated positive feedback to form an initial protrusive pattern and, later, to punctuate the PIP3 accumulation to maintain proper neurite outgrowth.

Molecular characterization of a transport vesicle protein Neurensin-2, a homologue of Neurensin-1, expressed in neural cells

We have isolated and characterized a novel cDNA encoding a small neuronal membrane protein showing high sequence homology to Neuro-p24/Neurensin-1, a protein containing a microtubule-associated domain at the carboxyl-terminus and exclusively localized to small vesicles of neurons. The newly identified Neurensin-2 constitutes two-membrane spanning domains but not the microtubule-binding domain, with a molecular mass of 28 kDa. Neurensin-2 mRNA is expressed only in brain, whereas the protein expressed in various neurons including those of the thalamus/hypothalamus and hippocampus, of postnatally developing mice. While the levels of Neurensin-1 mRNA and protein in retinoic acid-exposed mouse neuroblastoma Neuro2a cells increased, those of Neurensin-2 mRNA and protein remained unchanged. When the Neurensin-2 cDNA was transfected into Neuro2a cells, Neurensin-2 was expressed in small vesicles including lysosomes in the perinuclear region. On the cotransfection of Neurensin-1 and -2 cDNA into Neuro2a cells, Neurensin-2 was mainly found in small vesicles of the cell body and Neurensin-1 in those of growth cones. In nerve growth factor-stimulated PC12 cells, the intracellular localization of these proteins also differed. Furthermore, immunochemical staining of mouse brain revealed that Neurensin-1 and -2 had a similar distribution in many regions such as the Diagonal band, hippocampus, amygdaloid nucleus, and habenula nucleus, but differed in the intracellular localization as follows: Neurensin-1 was found mainly in neuritic processes, while Neurensin-2 was found in cell bodies. Thus, both Neurensin-1, and -2 are localized in small vesicles in neural cells, but their localizations of the vesicles are not always the same by each other, suggesting that they are under separate regulation.

A Bex‐cycle built for two

Neurotrophins promote neuronal survival, but it has been recognized that they also stimulate differentiation. Indeed, in early studies by Hamburger, Levi‐Montelcini and Cohen, nerve growth factor (NGF) not only promoted survival, but also caused marked neurite outgrowth when added to explants of sensory ganglia (Cohen et al , 1954). The ability of NGF to promote differentiation was particularly well illustrated when Greene & Tischler (1976) showed that a pheochromocytoma cell line (PC12) treated with NGF exited the cell cycle and differentiated into a sympathetic neuron‐like phenotype. Since then, it has been established that the survival and differentiation effects of the neurotrophins are primarily mediated by binding to members of the Trk family of tyrosine kinase receptors (Huang & Reichardt, 2003); however, the first receptor identified for NGF was a 75‐kDa protein referred to as p75 (Chao et al , 1986; Radeke et al , 1987). In the past few years, the p75 receptor has received a lot of attention because of its multifunctional signalling ability. This receptor can promote cell survival or induce apoptosis, regulate neurite outgrowth and promote peripheral myelination, depending on the specific cellular context, the expression of co‐receptors and the activating ligand (Barker, 2004). The role of p75 in promoting differentiation and cell‐cycle arrest has been largely overlooked in recent years, even though this was one of the first cellular responses to be shown to result from the binding of NGF to the receptor. Long considered to be simply a co‐receptor for the Trks, p75 came to be recognized as a bona fide signalling molecule when it was shown to activate sphingomyelinase. This resulted in the production of ceramide, which led to the differentiation of T9 glioma cells as indicated by their cell‐cycle arrest and process outgrowth (Dobrowsky et al , 1994). Although the focus of most subsequent research …

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.

The role of putative intragenic control elements in c-fos regulation by calcium and growth factor signalling pathways.

Sequences in the transcribed region of the c-fos gene have been suggested to control c-fos induction following exposure of cells to mitogens or stimuli that increase intracellular calcium concentrations. Using a mutational analysis we show that putative regulatory elements present in the first intron of the human c-fos gene and the fos-intragenic-regulatory-element (FIRE) are not required for c-fos regulation by growth factor and calcium signalling pathways in AtT20 and PC12 cells. Removal of the c-fos first intron and the FIRE did not increase the basal level of c-fos mRNA and only moderately reduced the magnitude of calcium-induced transcription mediated by either the entire c-fos promoter or the cAMP response element (CRE). Intragenic mutations did not affect serum response element (SRE)-dependent gene expression induced by calcium signals but caused a superinduction of c-fos expression in nerve growth factor-stimulated PC12 cells. These results indicate that c-fos promoter elements, rather than intragenic sequences, are the principal targets of transcription-regulating signalling pathways. This suggests that CRE- and SRE-bound activators of transcription initiation may also enhance, in a signal-dependent manner, c-fos transcript elongation beyond promoter-proximal pause sites.

Rapid lamellipodia formation in nerve growth factor-stimulated PC12 cells is dependent on Rac and PI3K activity

Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) but not by epidermal growth factor (EGF). However, features of differentiation such as neurite outgrowth are observable at the earliest after several hours. Using actin staining of the cells, we show here that NGF stimulation leads to lamellipodia formation within only 3 min at the periphery of the PC12 cells. EGF stimulation or microinjection of differentiation-inducing c-Crk I protein does not cause lamellipodia. The actin reorganization after NGF stimulation is blocked by microinjecting dominant negative Rac protein. The lamellipodia formation is also abolished by inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002 in a concentration-dependent manner. Phase-contrast time-lapse microscopy was used to analyze membrane dynamics in real time and to confirm the induction of lamellipodia by NGF and their inhibition by pretreatment with both wortmannin and LY 294002. The results indicate that NGF, but not EGF, leads to rapid lamellipodia formation in PC12 cells via phosphatidylinositol 3-kinase and the small GTPase Rac, thereby defining a novel role for these factors in early NGF signaling.

The Leukemia-associated Protein Btg1 and the p53-regulated Protein Btg2 Interact with the Homeoprotein Hoxb9 and Enhance Its Transcriptional Activation

BTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9.Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9.Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function.

Transcription Factor Phosphorylation by pp90rsk2: IDENTIFICATION OF Fos KINASE AND NGFI-B KINASE I AS pp90rsk2

The in vitro phosphorylation of transcription factors by growth factor-activated protein kinases has resulted in the discovery of a number of activities whose identities and relationships to one another are unclear. Fos kinase is a growth factor-stimulated serine/threonine protein kinase that phosphorylates c-Fos at serine 362 within the carboxyl-terminal regulatory domain. Fos kinase activation is dependent on p21(ras) and mitogen-activated protein kinase/ERK kinase kinase (MEK) activity and is independent of phosphatidylinositol 3-kinase activity. We have purified Fos kinase by affinity chromatography using the Sepharose-linked protein kinase inhibitor, bisindolylmaleimide (BIM). Fos kinase has an apparent molecular mass of 88 kDa, and mass spectrophotometric analysis of the isolated protein showed that it produced tryptic fragments identical to those predicted for pp90(rsk2). Fos kinase isolated from nerve growth factor-stimulated PC12 cells is indistinguishable from NGFI-B kinase I, based on their chromatographic behavior, substrate specificities, and relative sensitivity to BIM. Furthermore, we have distinguished Fos kinase from calcium/cAMP response element-binding protein (CREB) kinase. Therefore, Fos kinase and NGFI-B kinase I and pp90(rsk2) represent the same protein kinase species. Moreover, we report that pp90(rsk2) exists within nerve growth factor-stimulated PC12 cells as two chromatographically and immunologically distinct species. Finally, we demonstrate that CREB kinase is distinct from pp90(rsk2).

Inhibitors of p38 mitogen-activated protein kinase promote neuronal survival in vitro.

Mammalian mitogen-activated protein kinases include the extracellular signal-regulated protein kinase, the c-Jun amino-terminal kinase, and the p38 subgroups. Sustained activation of Jun kinase and p38 have been shown to precede apoptosis of PC12 pheochromocytoma cells induced by withdrawal of trophic factors. To investigate the possible role of p38 in neuronal apoptosis, we tested the effect of two selective p38 inhibitors, the pyridinyl imidazole compounds SB203580 and SB202190, on different populations of chick embryonic neurons in vitro. Both substances promoted the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent fashion. When assayed in nerve growth factor-stimulated PC12 cells, SB203580 pretreatment inhibited the activation of both ribosomal S6 kinases-1 and -2 with the same IC50 (approximately 30 microM) that inhibited apoptosis in primary neurons. Thus, p38 inhibitor-sensitive pathways may be involved in apoptosis of neurotrophic factor-deprived primary neurons, and in activation of ribosomal S6 kinases.

Inhibitors of p38 mitogen‐activated protein kinase promote neuronal survival in vitro

Mammalian mitogen-activated protein kinases include the extracellular signal-regulated protein kinase, the c-Jun amino-terminal kinase, and the p38 subgroups. Sustained activation of Jun kinase and p38 have been shown to precede apoptosis of PC12 pheochromocytoma cells induced by withdrawal of trophic factors. To investigate the possible role of p38 in neuronal apoptosis, we tested the effect of two selective p38 inhibitors, the pyridinyl imidazole compounds SB203580 and SB202190, on different populations of chick embryonic neurons in vitro. Both substances promoted the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent fashion. When assayed in nerve growth factor-stimulated PC12 cells, SB203580 pretreatment inhibited the activation of both ribosomal S6 kinases-1 and -2 with the same IC50 (approximately 30 μM) that inhibited apoptosis in primary neurons. Thus, p38 inhibitor-sensitive pathways may be involved in apoptosis of neurotrophic factor-deprived primary neurons, and in activation of ribosomal S6 kinases. J. Neurosci. Res. 52:483–490, 1998. © 1998 Wiley-Liss, Inc.