F11 Cells Research Articles

350 Characterization of endogenous PKC-Epsilon in F11 cell, a model system for studying pain signalling

350 Characterization of endogenous PKC-Epsilon in F11 cell, a model system for studying pain signalling

Extracellular ATP-dependent upregulation of the transcription cofactor LMO4 promotes neuron survival from hypoxia

Extracellular ATP is elevated by transient ischemia and is a potent signaling molecule in the central nervous system. ATP promotes neuron survival from serum starvation by activating P2Y purinergic receptors. ATP also activates IL-6 production and phosphorylation of Stat3 that promotes neuron survival. The transcription cofactor LMO4 is a positive mediator of IL-6/Stat3 signaling. Here, we found that LMO4 and the pro-survival factor cIAP2 (cellular inhibitor of apoptosis protein 2) are rapidly upregulated in neurons exposed to elevated extracellular ATP. Blocking LMO4 upregulation using siRNA in F11 cells blunted cIAP2 upregulation and abolished the early protective effect of ATP. Similar results were obtained using primary cortical neurons from LMO4 null mice, suggesting that LMO4 is required for ATP to protect neurons from hypoxia-induced apoptosis. Whereas increased Stat3 phosphorylation occurs after LMO4 and cIAP2 induction, the rapid upregulated phosphorylation of ERK and CREB may account for increased LMO4 and cIAP2 by ATP. ATP signaling through ERK and CREB activated LMO4 promoters and ERK activation increased LMO4 protein stability in F11 cells. Taken together, our studies reveal that LMO4 is a rapidly induced downstream effector of ATP signaling that promotes neuron survival from hypoxia.

LMO4 mRNA stability is regulated by extracellular ATP in F11 cells

LIM only domain protein 4 (LMO4) interacts with many signaling and transcription factors to regulate cellular proliferation, differentiation and plasticity. In Drosophila, mutations in the 3′ untranslated region (UTR) of the homologue dLMO cause a gain of function by increasing mRNA stability. LMO4 3′UTR contains several AU-rich elements (ARE) and is highly conserved among vertebrates, suggesting that RNA destabilizing mechanisms are evolutionarily conserved. Here, we found that extracellular ATP stabilized LMO4 mRNA in F11 cells. The LMO4 3′UTR added to a luciferase reporter markedly reduced reporter activity under basal conditions, but increased activity with ATP treatment. Two ARE motifs were characterized in the LMO4 3′UTR. ATP increased binding of HuD protein to ARE1. ARE1 conferred ATP and HuD-dependent mRNA stabilization. In contrast, sequences flanking ARE2 bound CUGBP1 and ATP destabilized this complex. Thus, our results suggest that ATP modulates recruitment of RNA-binding proteins to the 3′UTR to stabilize LMO4 mRNA.

TRPV1 at nerve endings regulates growth cone morphology and movement through cytoskeleton reorganization

While the importance of Ca(2+) channel activity in axonal path finding is established, the underlying mechanisms are not clear. Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones. These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N-arachidonoyl-dopamine (NADA), and affect the integrity of microtubule cytoskeleton. Using TRPV1-transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retraction, and collapse and formation of varicosities along neurites. These changes were due to TRPV1-activation-mediated disassembly of microtubules and are partly Ca(2+)-independent. Prolonged activation with very low doses (1 nM) of NADA results in shortening of neurites in the majority of isolectin B4-positive dorsal root ganglia neurones. We postulate that TRPV1 activation plays an inhibitory role in sensory neuronal extension and motility by regulating the disassembly of microtubules. This might have a role in the chronification of pain.

Pituitary adenylate cyclase-activating peptide (PACAP) induces differentiation in the neuronal F11 cell line through a PKA-dependent pathway

PACAP is a peptide with neuroprotective activity, which induces adenylate cyclase and protein kinase A (PKA) activity. PACAP has also been shown to induce neurite outgrowth in PC12 cells and dorsal root ganglion (DRG) neurons. Here, we report that exogenous PACAP38 promotes neurite outgrowth in the F11 neuroblastoma/dorsal DRG hybrid cell line. Using an automated microscopy system, we show that PACAP38 induces a 170-fold increase in neurite length, with an EC 50 of 3.1 nM, compared to 3.7 μM for forskolin and 143.4 μM for dibutyril cyclic AMP (dbcAMP). PACAP38 induced a 4-fold increase in the level of phosphorylation of cAMP-responsive element binding protein (CREB) in F11 cells with an EC 50 of 130 pM. In contrast a peptide related to PACAP, vasoactive intestinal peptide (VIP) failed to induce CREB phosphorylation or neurite outgrowth in F11 cells. Addition of the nonselective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX) increased the potency of PACAP at inducing neurite outgrowth by ten-fold. The PKA inhibitor, H89, was a potent inhibitor of PACAP38-induced neurite outgrowth. The delta-opioid receptor agonist, SNC 80, did not inhibit PACAP-induced neurogenesis even though it did reduce CREB phosphorylation. In contrast to previous studies in PC12 cells, PACAP38 failed to show MEK1 activation in F11 cells. PACAP is upregulated in DRG neurons as a result of injury, and F11 cells provide an easily accessible in vitro model for understanding mechanisms underlying PACAP differentiation and neurogenesis.

Validation of DRG-Like F11 Cells for Evaluation of KCNQ/M-Channel Modulators

F11 cells are derived from a fusion between mouse embryonic neuroblastoma and rat dorsal root ganglion (DRG) neurons. These cells have been shown to retain many features of native DRG neurons, including synthesis of neurotransmitters, expression of neuropeptide receptors, and voltage-gated calcium channels. In this study, we describe the presence of KCNQ2/3 channels in F11 cells as determined by both reverse transcription-polymerase chain reaction and functional assessment. Electrophysiological recordings in whole-cell configuration performed in F11 cells revealed the functional expression of a KCNQ/M-current with characteristic slow deactivation kinetics, similar to the KCNQ/M-current recorded from dissociated DRG neurons. Deactivation tail currents elicited by conventional M-current protocols were enhanced by a specific KCNQ/M-channel opener, WAY-1, and inhibited by the specific blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)- anthracenone]. Using a non-radioactive atomic absorption Rb+ efflux assay, we further validated that Rb+ efflux can be induced in differentiated F11 cells by activation of KCNQ/M-channels. These findings have led us to conclude that F11 cells can be used as a DRG cell model to evaluate effects of KCNQ/M-channel modulators.

Rapid disassembly of dynamic microtubules upon activation of the capsaicin receptor TRPV1

The transmission of pain signalling involves the cytoskeleton, but mechanistically this is poorly understood. We recently demonstrated that the capsaicin receptor TRPV1, a non-selective cation channel expressed by nociceptors that is capable of detecting multiple pain-producing stimuli, directly interacts with the tubulin cytoskeleton. We hypothesized that the tubulin cytoskeleton is a downstream effector of TRPV1 activation. Here we show that activation of TRPV1 results in the rapid disassembly of microtubules, but not of the actin or neurofilament cytoskeletons. TRPV1 activation mainly affects dynamic microtubules that contain tyrosinated tubulins, whereas stable microtubules are apparently unaffected. The C-terminal fragment of TRPV1 exerts a stabilizing effect on microtubules when over-expressed in F11 cells. These findings suggest that TRPV1 activation may contribute to cytoskeleton remodelling and so influence nociception.

Involvement of tyrosine kinases and STAT3 in Humanin-mediated neuroprotection

Humanin (HN) inhibits neuronal cell death induced by various Alzheimer's disease (AD)-related insults. It has been proposed that HN binds to a putative receptor on the cell membrane and triggers a signal transduction cascade linked to neuroprotection. Recently, it was shown that HN binds to pertussis toxin (PTX)-sensitive G protein-coupled formylpeptide receptor-like-1 molecule (FPRL-1), reduces Aβ(1-42) aggregation and fibril formation, and suppresses the Aβ(1-42) toxicity on mononuclear phagocytic cells [Ying, G., Iribarren, P., Zhou, Y., Gong, W., Zhang, N., Yu, Z.X., Le, Y., Cui, Y., Wang, J.M., 2004. Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor. Journal of Immunology 172 (11), 7078–7085.]. We here show that siRNA-mediated disruption of expression of the mouse counterpart of FPRL-1, FPR2, did not result in attenuation of HN-mediated rescue of neuronal cell death induced by AD-related insults. We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases. Altogether, we speculate that a receptor other than FPR2 exists that mediates HN neuroprotection in F11 neurohybrid cells

In vitro and in vivo effects of an α3 neuronal nicotinic acetylcholine receptor antisense oligonucleotide

In the mammalian central nervous system (CNS), a family of α and β subunits (α2-7, β2-4) assemble to form both hetero- and homopentameric neuronal nicotinic acetylcholine receptors (nAChRs). In contrast to α4β2 and α7, the predominant brain subtypes, far less is known regarding the functional expression and significance of α3-containing nAChRs in the CNS. In trying to better understand the role α3 in the CNS, an antisense knockdown strategy was utilized in the present studies. Specifically, Isis 106567 was identified out of 80 antisense oligonucleotides (aONs) designed and screened for their ability to reduce α3 mRNA expression in PC-12 cells. In addition to reducing α3 mRNA by greater than 75%, Isis 106567 attenuated nicotine-induced calcium influx in α3-expressing F11 cells. In vivo studies revealed significant reduction of α3 mRNA levels in both thalamus and medial habenula, regions known to express α3, following continuous (7 days) intracerebroventricular (i.c.v.) infusion of Isis 106567 in rats. Consistent with functional α3 knockdown, epibatidine-induced c-Fos expression in the medial habenula was attenuated in aON-treated rats. Known physiological responses elicited by epibatidine, such as hypothermia and micturition, were not affected by α3 aON treatment. However, the incidence of epibatidine-induced seizures was reduced in α3-antisense aON-treated rats, suggesting that α3 may be involved in mediating seizures produced by the nAChR agonist. Results of our studies suggest that Isis 106567 may be a useful in vivo tool for characterizing the functional significance of α3 expression in the CNS.

Nonspecific association of 2',3'-cyclic nucleotide 3'-phosphodiesterase with the rat forebrain postsynaptic density fraction.

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.

Modulation of the N-type calcium channel gene expression by the α subunit of Go

Go, a heterotrimeric G-protein, is enriched in brain and neuronal growth cones. Although several reports suggest that Go may be involved in modulation of neuronal differentiation, the precise role of Go is not clear. To investigate the function of Go in neuronal differentiation, we determined the effect of Goα, the α subunit of Go, on the expression of Ca v2.2 , the pore-forming unit of N-type calcium channels, at the transcription level. Treatment with cyclic AMP (cAMP), which triggers neurite outgrowth in neuroblastoma F11 cells, increased the mRNA level and the promoter activity of the Ca v2.2 gene. Overexpression of Goα inhibited neurite extension in F11 cells and simultaneously repressed the stimulatory effect of cAMP on the Ca v2.2 gene expression to the basal level. Targeted mutation of the Goα gene also increased the level of Ca v2.2 in the brain. These results suggest that Go may regulate neuronal differentiation through modulation of gene expression of target genes such as N-type calcium channels .

Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells.

Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor expressed in neuronal precursors in the developing nervous system. The function of Ngn1 in neurogenesis has been shown in various aspects. In this study, we investigated the neurogenic potential of Ngn1 using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To investigate the expression of Ngn1, expression vectors for the full-length and the C- terminal deletion mutant of Ngn1 were constructed and their transactivation potential was verified using reporter gene containing the E-box sequence. Overexpression of the full-length Ngn1 induced neurite outgrowth in F11 cells in the absence of cAMP. A C-terminal deletion mutant, Ngn1(1-197), inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that the Ngn1 plays an important role in differentiation of neuroblastoma cells and the C terminus of Ngn1 is essential for the efficient differentiation.

A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels.

The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 +/- 6.8 % 5-HT-mediated, but not GTP-gamma-S-induced, inhibition of Ca(2+) current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP-gamma-S. 5-HT(1A) receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT(1A) receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca(2+) channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT(1A) receptors, with only a 13.4 +/- 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT(1A) receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-5-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT(1A) receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca(2+) channel.

Overexpression of BETA2/NeuroD induces neurite outgrowth in F11 neuroblastoma cells

BETA2/NeuroD, a basic helix‐loop‐helix (bHLH) transcription factor, has been shown to play important roles in the development of the nervous system and the maintenance and formation of pancreatic and enteroendocrine cells. The gain of function of BETA2/NeuroD in neurogenesis has been shown in Xenopus embryos. In this study, we investigated the neurogenic potential of BETA2/NeuroD using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To induce or block the expression of BETA2/NeuroD, expression vectors for the full‐length and a C‐terminal deletion mutant of BETA2 were constructed and their transactivation potential was verified using reporter genes containing the insulin promoter sequences. Overexpression of BETA2 with full‐length construct induced neurite outgrowth in F11 cells in the absence of cAMP. In contrast, the C‐terminal deletion mutant, BETA2(1–233), which has dominant negative activity, inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that BETA2/NeuroD plays an important role in terminal differentiation of neuroblastoma cells. They also imply that BETA2/NeuroD or related bHLH factors plays an essential role for differentiation of F11 neuroblastoma cells.

Overexpression of BETA2/NeuroD induces neurite outgrowth in F11 neuroblastoma cells.

BETA2/NeuroD, a basic helix-loop-helix (bHLH) transcription factor, has been shown to play important roles in the development of the nervous system and the maintenance and formation of pancreatic and enteroendocrine cells. The gain of function of BETA2/NeuroD in neurogenesis has been shown in Xenopus embryos. In this study, we investigated the neurogenic potential of BETA2/NeuroD using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To induce or block the expression of BETA2/NeuroD, expression vectors for the full-length and a C-terminal deletion mutant of BETA2 were constructed and their transactivation potential was verified using reporter genes containing the insulin promoter sequences. Overexpression of BETA2 with full-length construct induced neurite outgrowth in F11 cells in the absence of cAMP. In contrast, the C-terminal deletion mutant, BETA2(1--233), which has dominant negative activity, inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that BETA2/NeuroD plays an important role in terminal differentiation of neuroblastoma cells. They also imply that BETA2/NeuroD or related bHLH factors plays an essential role for differentiation of F11 neuroblastoma cells.

Kinetics of tumorigenic vascular endothelial growth factor signalling and its significance in human hepatocellular carcinoma cells

Ras-VEGF-concerned angiogenesis is correlated with oncogene maintenance, tumorigenesis, metastasis and resistance to anti-cancer therapies; however, this association is not clearly elucidated by serum VEGF, due to VEGF signalling in blood cells themselves. The present study aimed to elucidate tumorigenic VEGF signalling in eight human HCC cell types and reveal the kinetics of tumorigenic VEGF signalling in three time intervals, thereby discovering the relationships of VEGF-concerned angiogenesis signalling with the extent of the human HCC cell growth, metastasis and resistance to anti-cancer drugs, by using the poorly metastatic SMMC7721, 7402/D + (doxorubicin-resistance) and 7402/D – (doxorubicin-withdrawal), the highly metastatic MHCC1 non-transfected human HCC cell lines, and the highly metastatic A3-1, F8, F11 and E3 human HCC cell lines transfected with expressing green fluorescence protein into the phenotype of MHCC1 cells, and quantitative ‘sandwich’ ELISA analyses. The unique results indicated attributes and objective laws as follows. Human HCC cell growth requires time-dependent tumorigenic VEGF signalling; levels of VEGF signalling are positively correlated with each cell phenotype itself; and levels of VEGF signalling are inversely correlated with the possibility of metastasis and drug resistance. The contrast data first reveal important clues for exploring dual metastatic mechanisms via tumor cell-generated non-endothelium vasculogenesis and VEGF-endothelium-attached angiogenesis that may be essential for developing novel strategies aimed at VEGF-concerned signal networks in ischemic/metastatic diseases and transgenic models.

Antibody-Regulated Neurotoxic Function of Cell-Surface β-Amyloid Precursor Protein

APP is a transmembrane precursor of β-amyloid, and its mutations cause early-onset familial Alzheimer's disease. We report a toxic function of normal wild-type APP (wtAPP). Treatment of neuronal F11 cells, immortalized embryonic day 13 neurons, overexpressing wtAPP with anti-APP antibodies caused death. Death was not induced by antibody in parental F11 cells. Death by antibody occurred through cell-surface APP, not through secreted APP, in a pertussis toxin-sensitive manner and was typical apoptosis, not observed in primary astrocytes or glioma cells overexpressing wtAPP, but observed in primary cortical neurons. Cell-surface APP thus performs a toxic function as an extracellularly controllable regulator of neuronal death. This study provides a novel insight into the normal and pathological functions of cell-surface wtAPP.

Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the alzheimer betaA4-amyloid precursorprotein.

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.

Neuronal Apoptosis by Apolipoprotein E4 through Low-Density Lipoprotein Receptor-Related Protein and Heterotrimeric GTPases

The epsilon4 genotype of apolipoprotein E (apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the apoE4 protein (ApoE4) evokes apoptosis in neuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 microg/ml ApoE4, but not by 30 microg/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, alpha2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Galpha(i) restored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G(i) class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.

Neurite outgrowth induced by cyclic AMP can be modulated by the alpha subunit of Go.

Although abundant Go has been found in nervous tissues and it has been implicated in neuronal differentiation, the mechanism of how Go modulates neuronal differentiation has not been defined. Here, we report that the alpha subunit of Go (alphao) modulates neurite outgrowth by interfering with the signaling pathway initiated by cyclic AMP (cAMP). In F11 cells, cAMP induced neurite outgrowth and activated cAMP-responsive element binding protein (CREB). Specific inhibition of cAMP-dependent protein kinase reduced both CREB activity and neurite outgrowth (NOG). Interestingly, cAMP reduced phosphorylation of extracellular signal-regulated kinase (Erk). Neither a dominant negative form nor an active form of Ras altered neurite outgrowth. Expression of alphao (alphao(wt)) decreased the average length of neurites but increased the number of neurites per cell. An active mutant, alphaoQ205L, which lost GTPase activity and thus could not bind to Gbetagamma, gave similar results, suggesting that the effect of alphao is not mediated through Gbetagamma. Expression of ao(wt) or alphaoQ205L also prohibited CREB activation. Thus, activation of Erk may not be essential for neuronal differentiation in F11 cells and alphao may cause changes in NOG by inhibiting CREB activation.