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Section on Molecular Neuroscience (A.R., D.V., M.J.G., L.E.E.) and Section on Directed Gene Transfer (M.V.E.), Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland; and Institut National de la Santéet de la Recherche Médicale U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (l'Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), University of Rouen, Mont-Saint-Aignan, France (A.R., D.V., A.F.-M., B.J.G., H.V.)
Received January 2, 2008; accepted March 17, 2008
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Abstract |
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; Vaudry et al., 2002b), although other effects of NGF, such as induction of sodium channel expression, do require cAMP (Ginty et al., 1992; Yao et al., 1998). A significant literature also implicates cAMP in a broad range of neuronal differentiation responses, including neuritogenesis, survival, regeneration, repair, and expression of genes encoding neuron-specific proteins, such as neurotransmitter biosynthetic enzymes, neuropeptides, receptors, and ion channels (Qiu et al., 2002), although the effects of first messengers that regulate cAMP generation in differentiating neurons have not been studied as extensively as receptor tyrosine kinase-stimulating neurotrophins such as NGF. The neuropeptide PACAP has garnered significant interest in this regard, because PACAP is neuritogenic upon exposure to PC12 cells (Deutsch and Sun, 1992), enhances neuronal survival of cerebellar granule cells (Kienlen Campard et al., 1997) and PC12 cells (Tanaka et al., 1996), and is neuroprotective after ischemic injury to the brain (Reglodi et al., 2000; Chen et al., 2006; Ohtaki et al., 2006).
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). A number of transcripts induced by PACAP in PC12 cells are both ERK-dependent (blocked by U0126) and insensitive to PKA inhibition (by H89) (Vaudry et al., 2002a) and some of these are also induced by forskolin even in the presence of H89 (Gerdin and Eiden, 2007). We therefore hypothesized that PACAP signaling to ERK to initiate neurite formation might proceed through a noncanonical (PKA-independent) cAMP-dependent signaling pathway. In pursuit of this hypothesis, we screened PACAP target genes in PC12 cells also induced by forskolin and dibutyryl cAMP whose regulation is both ERK-dependent and independent of activation of protein kinase A, and whose transcription might be required for various aspects of differentiation induced by PACAP.
Rap1 is a potential regulator of ERK that can be activated by cAMP through both PKA-dependent and -independent pathways (York et al., 1998; Gerdin and Eiden, 2007). Maximal activation of total cellular Rap1 by PACAP in PC12 cells requires the participation of a number of protein kinases (Bouschet et al., 2003), and a Src-dependent activation of Rap1 initiated by cAMP and mediated by PKA has been identified in PC12 cells (Obara et al., 2004). However, the functional significance of Rap1 activation by each of these pathways, particularly for neuritogenesis, has not yet been addressed. Thus it is established in PC12 cells that PACAP elevates cAMP; that cAMP can activate Rap1; that Rap1 activation can persistently stimulate total cellular ERK; and that constitutively active ERK can drive neuritogenesis (Deutsch and Sun, 1992; Vossler et al., 1997; Yao et al., 1998; York et al., 1998; Harada et al., 2001; Stessin et al., 2006). However, a coherent signaling mechanism underlying PACAP-induced PC12 cell differentiation remains to be elucidated. Here, we address two key questions toward this end. First, which PACAP-initiated differentiating responses of PC12 cells are mimicked by cAMP, and which of these require PKA? Second, does PACAP activate a cohort of genes in PC12 cells that are also activated by elevation of cAMP alone, and does abrogation of expression of any of these transcripts affect the PACAP-induced functional differentiative responses of neuritogenesis, increased cell size, and cessation of proliferation? The experimental answers to these questions provide a mechanism for PACAP-induced neuritogenesis involving cAMP-initiated, PKA-independent activation of ERK, and subsequent expression of specific genes that drive distinct components of the differentiation program. These signaling mechanisms may also be relevant for cAMP-dependent signaling for differentiation by first messengers acting through other G-protein coupled receptors besides the PACl receptor activated by PACAP in PC12 cells.
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Materials and Methods |
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) was grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 7% heat-inactivated fetal bovine serum (Sigma), 7% horse serum (Lonza Bioscience, Walkersville, MD), 2.5% HEPES (Invitrogen), 1% glutamine (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37°C in a 10% CO2/90% air humidified atmosphere. Two days before treatment, cells were replated on poly-L-lysine-coated Petri dishes (Costar; Corning Life Sciences, Acton, MA). When used, inhibitors were added 30 min before exposure to PACAP, forskolin, or dbcAMP. Quantitative Analysis of Neurite Outgrowth. Two days after treatment, images of PC12 cells were randomly acquired on a computer-assisted microscope [IPLab from BD Biosciences Bioimaging (Rockville, MD) and Metamorph from Molecular Devices, (Sunnyvale, CA)]. Differentiation was investigated on more than 22,000 cells by measuring neurite length. The percentage of cells bearing neurites was quantified, the number of neurites per cell was counted, and the total neurite outgrowth for each cell was measured. Neurites were defined as cell processes greater than 6 µm, to eliminate inadvertent counting of cell membrane ruffling or irregularities as neurites.
Quantification of Cell Number and Measurement of Cell Size. Two days after treatment, cells were washed with phosphate-buffered saline and detached by incubation with Accutase (Innovative Cell Technologies, La Jolla, CA) at 37°C for 15 min. Cell size and number were measured with a cell counter (Z2; Beckman Coulter, Fullerton, CA) with lower and upper limits set to 10 and 17 µm, respectively. Preliminary experiments demonstrated that a 17-µm cutoff on the cell-counting instrument (above) provided the most sensitive and reliable indicator of changes in PC12 cell volume after treatment for two days with PACAP (100 nM). A dose response with graded concentrations of PACAP and VIP (10 pM-1 µM) confirmed that a 17-µm cutoff provided results that correlated well with a direct measurement of the cell diameter (Fig. 1C).
cAMP Quantification. 30 min after treatment, cAMP production was quantified with a [3H]cAMP assay kit (Amersham, Chalfont St. Giles, Buckinghamshire, UK) as described previously (Hamelink et al., 2002).
Western Blot Analysis. Proteins contained in PC12 cells were extracted in lysis buffer consisting of 1% Triton X-100, 50 mM Tris-HCl, and 10 mM EDTA. The homogenate was centrifuged (14,000g, 4°C, 15 min), and proteins contained in the supernatant were precipitated at 4°C by addition of ice-cold 10% trichloroacetic acid. The extract was centrifuged (12,000g, 4°C, 15 min) and washed three times with ether/alcohol [70:30 (v/v)]. The pellet was denatured in 50 mM Tris-HCl, pH 7.5, containing 20% glycerol, 0.7 M 2-β-mercaptoethanol, 0.002% (w/v) bromphenol blue and 3% (w/v) SDS at 100°C for 5 min, and electrophoresed on a 10% SDS-PAGE gel. After separation, proteins were electrically transferred onto a nitrocellulose membrane (Amersham). The membrane was incubated with blocking solution (0.5% bovine serum albumin and 2% milk in Tris-buffered saline containing 0.05% Tween 20) at room temperature for 1 h, and developed with antibodies against phosphorylated and total cellular ERK (Promega) using a chemiluminescence detection kit (ECL System; Amersham). Autoradiographic films were quantified using an image analysis system (Biocom, Les Ulis, France).
Rap1 Activation Assay. Fusion protein GST-Ral-RBD produced in Escherichia coli in the presence of isopropyl β-D-thiogalactopyranoside was a generous gift from Dr. Michel Philippe (Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6187, University of Poitiers, France). The bacterial pellet was suspended in sodium-Tris-EDTA buffer (10 mM Tris, pH 8.0, 150 mM NaCl, and 1 mM EDTA) in the presence of 1 mg/ml lysozyme, protease inhibitors (10 µg/ml trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), 1 mM dithiothreitol, and 1.5% N-lauryl sarcosyl. The protein was affinity purified by incubation at 4°C overnight with glutathione Sepharose 4B beads.
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Rap1b Dominant-Negative Vector. Rap1b DN (S17N) cloned into pcDNA 3.1(+) was a generous gift from Dr. Elisabeth Bock (Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Denmark). The Rap1b DN insert was first subcloned into the pIRES2-eGFP vector (Clontech, Mountain View, CA) and the Rap1b DN-IRES-eGFP was subsequently cloned upstream of eGFP into the lentivirus packageable genome pRRLsin.CMV.eGFP-wpre as an XbaI-BamHI fragment.
Viral particles were generated by transient cotransfection of the packageable genome with gag/pol and vesicular stomatitis virus envelope expression plasmids in the 293T cell line. Forty-eight hours later, the culture medium containing the viral particles was collected, filtered, and added to PC12 cells. After overnight exposure to the viral particles, cells were washed twice with fresh medium. Forty-eight hours later, Rap1b DN-IRES-eGFP infected cells stably expressing the transduced proteins were identified under a fluorescent microscope. Cells transduced with a pRRLsin.CMV.GFPpre vector were used as a control.
RNA Isolation, Microarray Experiments, and Data Analysis. After 6 h of treatment, total RNA was extracted with TRIzol reagent (Invitrogen) and further purified with the RNeasy Mini Kit (QIAGEN, Valencia, CA). The RNA concentration was measured by absorbance at 260 nm, and RNA integrity was confirmed by denaturing gel electrophoresis.
The cDNA sequences used in this study were issued from the NIA Mouse 15K cDNA clone set (see http://lgsun.grc.nia.nih.gov/cDNA/15k.html for details). PCR products generated from these clones were printed onto polylysine-coated glass slides at the National Human Genome Research Institute microarray facility. Fluorescence-labeled cDNA was synthesized from 10 µg of RNA from treated or untreated PC12 cells, with the SuperScript First Strand Synthesis System for RT-PCR (Invitrogen) in the presence of amine-modified random primers and aminoallyl-dUTP/dNTP. Probes were then labeled with N-hydroxy-succinimide ester dye Cy3 or Cy5 (Amersham). After denaturation, purified Cy3/Cy5-labeled cDNA samples were combined and hybridized on a microarray slide in a humidified chamber (Corning Life Sciences) at 65°C overnight in the presence of 5x saline-sodium citrate (SSC), 0.1% SDS, 25% formamide and polyA (25 ng/µl). Before scanning at 532 nm for Cy3 and 633 nm for Cy5 (Agilent Technologies, Foster City, CA), slides were successively washed at room temperature in 0.5x SSC/0.1% SDS for 2 min, 0.5x SSC for 2 min (twice), and 0.06x SSC for 2 min. The two fluorescent images obtained from the scanner were analyzed using the IPLab software. The data from 37 successful experiments were entered into the FileMaker Pro 5 software (FileMaker, Santa Clara, CA) to cluster the genes regulated in the various experimental conditions and to conduct a functional analysis. Genes were included as induced by a given treatment if 1) all values in the data set had a quality index of >0.3 for the combined ratio value and 2) the mean induction value was >1.5 fold.
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Ct method according to Applied Biosystems instructions.
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siRNA Experiments. Transfection of siRNA into PC12 cells was performed with the Amaxa Nucleofector (Amaxa, Koeln, Germany) according to the instructions of the manufacturer. In brief, 2 x 106 cells were resuspended into 120 µl of Nucleofector solution containing 20 µg of siRNA. Immediately after electroporation, fresh medium was added, and cells were cultivated at 37°C in a 10% CO2/90% air incubator. Several siRNA were designed and tested to inhibit immediate early response 3 (Ier3), villin 2 (Vil2), and early growth response 1 (Egr1) gene expression (Hp flexible siRNA; QIAGEN). The sequences of the siRNA used for the experiments presented in this study were CAA CGC TAA CTC AGA ACA CTA for Ier3, AGC GAT AAT ATG GGT TTG TAA for Vil2, AAG GCG CTG GTG GAG ACA AGT for Egr1-siRNA1, ATT GTA CTA TTT GGA GTT AAA for Egr1-siRNA2, and CAA ACC AAT GGT GAT CCT CTA for Egr1-siRNA3.
The specificity of the effect of Egr1 siRNA on cell differentiation was confirmed using three different sequences (Egr1-siRNA1, 2 and 3), which all reduced Egr1 mRNA levels as well as PACAP-induced neuritogenesis. Egr1-siRNA1 was chosen for further work. The capacity of Egr1 siRNA1 to specifically reduce its cognate mRNA was confirmed by measuring its effect on induction of Egr1 mRNA, and three other mRNAs (Ier3, Odc, and Rgs2) by 100 nM PACAP. Egr1 siRNA1 decreased only the expression of its cognate mRNA target. Further testing of Egr1 and Vil2 siRNAs against their cognate mRNAs likewise revealed no off target effects of these siRNAs.
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Statistical Analysis. Data are presented as the mean ± S.E.M. from at least three independent experiments performed in triplicate, except for the histograms reporting the percentage of cells with a diameter >17 µm in Fig. 11, which are the mean ± S.E.M. from a representative experiment that was repeated 4 times. Unless otherwise stated, statistical analyses were conducted using a Kruskal-Wallis test, followed by Dunn's post tests or by the Mann-Whitney test using Prism software (GraphPad Software, San Diego, CA).
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). Differences in PACAP potency to stimulate neuritogenesis and to increase cell size or decrease proliferation suggest that these effects are mediated by distinct signaling pathways. The minimum time of exposure to PACAP required for a full PACAP response 48 h later, however, was similar for neuritogenesis and cell growth arrest (Fig. 2, A and B), indicating that 1 to 6 h was an appropriate temporal window for examining cellular transcriptional changes elicited by PACAP that are likely to underlie neuritogenesis, altered cell size, or cessation of cell growth.
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), and cAMP is therefore a prime candidate for the second messenger that effects changes in cell morphology and function. The cellular and biochemical profiles for PC12 cell differentiation induced by forskolin (25 µM), an adenylate cyclase stimulator, and dibutyryl cAMP (10 mM), which generates cAMP upon intracellular hydrolysis, were compared with that of 100 nM PACAP. All three agents induced neuritogenesis within 48 h of exposure that was unaffected by pretreatment with the protein kinase A inhibitor H89 and blocked by the MEK inhibitor U0126 (Fig. 3, A and B). The effect of PACAP and forskolin on cell size persisted in the presence of H89 (Fig. 3C). In contrast, the growth arrest activity of PACAP was significantly reduced, and that of forskolin totally abolished, in the presence of H89 (Fig. 3D). Finally, the adenylate cyclase inhibitor 2',5'-dideoxyadenosine inhibited PACAP-induced cAMP production (Fig. 4A) and significantly reduced the effect of PACAP on neurite outgrowth (Fig. 4, B and C) and cell proliferation (Fig. 4D).
Protein kinase C is often a major contributor to neurotrophin signaling, leading to neuroendocrine cell differentiation, including several of the differentiative effects of NGF on PC12 cells, such as neuritogenesis (Das et al., 2004). However, the specific PKC inhibitor chelerythrine did not block PACAP-induced neuritogenesis or growth arrest (Fig. 5). Likewise, the broad spectrum (protein kinases A and C) inhibitor H7 failed to block PACAP-induced neuritogenesis (Fig. 5D). These data provide further criteria to define PACAP target genes involved in neuritogenesis and changes in cell size based on their cellular and biochemical responses to kinase inhibition, and prompted us to focus on regulation through the cAMP pathway.
MAP Kinase Induction by PACAP Independent of PKA. The ERK MAP kinase pathway has been shown by others to be required for PACAP-induced neurite outgrowth in PC12 cells based on inhibition with the MEK inhibitor PD98059 (Barrie et al., 1997; Lazarovici et al., 1998). Western blot experiments confirmed that PACAP induced a rapid and strong phosphorylation of ERK without affecting total ERK (Fig. 6A). Furthermore, this action of PACAP was independent of PKA in that it was unaffected by H89 (Fig. 6A). Both U0126 and PD98059 blocked PACAP-induced ERK phosphorylation (data not shown) in parallel with blockade of PACAP-induced neuritogenesis (>60 and >75% reduction in number of neurites per cell and total neurite length, respectively; Fig. 6B). The effect of PACAP on the percentage of cells with a diameter above 17 µm was significantly reduced in the presence of U0126 (Fig. 6C), and there was no difference between cells treated with U0126 alone or PACAP plus U0126. The MEK inhibitor U0126 decreased cell number, as expected given the role of MAPK in cell proliferation in serum-containing medium, and thus the involvement of MAPK in PACAP signaling for growth arrest could not be reliably evaluated (Fig. 6D).
Rap1 Involvement in PACAP Signaling to Neuritogenesis. The effects of PACAP on both neurite outgrowth and ERK activation seem to be independent of either PKA or PKC (Figs. 3, 4, 5 and 6). Based on the fact that ERK phosphorylation has been shown to involve Rap1 activation (Bouschet al., 2003), a possible ERK-dependent regulation of neurite outgrowth through Rap1 was investigated. Exposure of PC12 cells to PACAP (100 nM) provoked a rapid and transient activation of Rap1, with a maximal increase observed after 30 s of treatment (Fig. 7A). The relatively small increase in total Rap1 activation observed may indicate that PACAP signaling reaches only a subcompartment of cellular Rap1 under our culture conditions, which do not include serum starvation before measurement of Rap activation.
PC12 cells transduced with a dominant-negative form of Rap1 (Rap-DN) coupled with an IRES-GFP showed no morphological differences from nontransduced cells, but after 48 h of treatment with PACAP (100 nM), Rap-DN expressing cells had fewer and shorter neurites than PC12 cells not expressing Rap-DN (Fig. 7B). Blocking Rap1 signaling decreased both the number of neurites per cell and the total neurite length after PACAP treatment without affecting the percentage of cells with neurites, suggesting that neurite outgrowth, rather than neurite initiation, is the component of neuritogenesis primarily affected by Rap1-dependent signaling (Fig. 7C). The expression of GFP alone in PC12 cells had no effect on PACAP-induced neurite outgrowth (data not shown).
PACAP, Forskolin, dbcAMP, and NGF Regulation of Both Common and Distinct Genes in PC12-G Cells. The PC12 cell transcriptome was investigated after 6 h of treatment with PACAP (100 nM), forskolin (25 µM), or dbcAMP (1 mM) in an attempt to identify cAMP-dependent target genes potentially involved in PACAP-induced neuritogenesis and increased cell size. Treatment with NGF (100 ng/ml) was used as a comparison: NGF induction of neuritogenesis is independent of cAMP (Vaudry et al., 2002b). Incubation of PC12 cells with PACAP for only 6 h was sufficient to elicit the later full-length neurite outgrowth and increase in cell size observed at 48 h (Fig. 2); therefore, this time was chosen for microarray analysis. Among the 15,000 cDNAs present on the microarray, 118, 64, 48, and 133 unique transcripts were significantly induced by PACAP, forskolin, dbcAMP, and NGF, respectively (Fig. 8, Tables 2, 3, 4 and 5). Twenty-seven of these transcripts were regulated in common by PACAP, forskolin, and dbcAMP (Table 2), 13 exclusively by PACAP and forskolin (Table 2), three only by dbcAMP and PACAP (Table 2), and eight only by forskolin and dbcAMP (Fig. 8A, Table 2). Comparison of the PACAP and NGF transcriptomes revealed that 19 transcripts were induced in common by the two neurotrophic factors (Fig. 8B, Table 2), and among these only three were also activated by forskolin and dbcAMP (Fig. 8C, Table 2). Other transcripts were only induced by PACAP (Table 3), forskolin (Table 4), dbcAMP (Table 4), or NGF (Table 5).
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To verify the microarray results, primers for Q-RT-PCR were designed against 17 transcripts with varying expression profiles in microarray analysis (Table 1). We chose six of the 17 genes for further analysis based on their up-regulation by all three cAMP-elevating or cAMP-mimicking agents (PACAP, dbcAMP, and forskolin), and 11 additional representative transcripts from other categories in which a 1.5-fold or greater increase was seen with only one. These 17 transcripts were validated in two ways. First, transcripts induced after 6 h of treatment with PACAP (100 nM), forskolin (25 µM), dbcAMP (10 mM), or NGF (100 ng/ml) as detected by microarray hybridization were also found to be elevated via quantification using real-time PCR (Table 6). Some transcripts, such as glutaredoxin (Glrx) detected as elevated only by PACAP in the microarray experiments, were in fact also induced by forskolin and dbcAMP when measured using real-time PCR (Table 6). Most transcripts [e.g., GATA binding protein 2 (Gata2), PACAP specific receptor-1 (Pac1), and Neuropilin (Nrp1)] that were not induced by PACAP according to microarray analysis were indeed not regulated as confirmed by real-time PCR (Table 6). A Q-RT-PCR time course (Fig. 9) was then carried out for all 17 transcripts to investigate the possibility of artifactual discordance in transcript regulation by PACAP, forskolin, dbcAMP, or NGF based solely on the decreased sensitivity of microarray analysis compared with Q-RT-PCR (Vaudry et al., 2002a). The time course confirmed that these transcripts showed a robust up-regulation by all four pharmacological (dbcAMP, forskolin) or neurotrophic (PACAP, NGF) neuritogenic agents during the first 48 h of treatment, at which time neuritogenesis is maximal for PACAP, dbcAMP, and forskolin and is well under way for NGF. Seven of 17 transcripts (Gata2, Nrp1, Pac-1, Anx2, Homer2, Akr1b8, and Glrx) failed to fulfill this second criterion. We chose three of the remaining ten transcripts (Egr1, Vil2, and Ier3) for further analysis based on the overall robustness of induction by all four agents over the first half of the 48-h time course (Fig. 9). Thus, the Q-RT-PCR time course experiment revealed that the transcript encoding early growth response 1 (Egr1; Fig. 9), which was found to be activated only by NGF at 6 h after microarray analysis, was induced earlier by cAMP and PACAP and returned to control levels after 6 h of treatment. The immediate early gene Ier3, initially thought to be differentially regulated by PACAP, forskolin, and dbcAMP versus NGF, was also shown to be transiently regulated by NGF, albeit considerably less (5-fold) than that by cAMP (maximally, 15-25-fold; Fig. 9). The transcript encoding villin 2 (Vil2; Fig. 9) was up-regulated modestly (2-4-fold) but consistently by all four agents with a maximum at around 3 h of treatment.
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Regulation of cAMP- and PACAP-Dependent Genes by PKA and ERK. The effects of PACAP on neuritogenesis and cell size seem to be initiated by cAMP and mediated through downstream signal transduction mechanisms that include ERK but not PKA, whereas PACAP-induced growth arrest includes a PKA-dependent, or at least an H89-inhibited, component (Figs. 3, 6). The involvement of PKA and ERK in the regulation of three prominent cAMP- and PACAP-dependent transcripts identified by microarray cluster analysis, namely Ier3, Egr1, and Vil2, were further investigated by Q-RT-PCR in the presence and absence of H89 (10 µM) or U0126 (25 µM; Fig. 10). Ier3 induction was only slightly reduced in the presence of H89 but was strongly inhibited by U0126. Egr1 induction by PACAP was unaffected by H89 but completely blocked by U0126. Induction of Vil2 by PACAP was, like cell size, unaffected by H89 and blocked, but to a lesser degree than either neuritogenesis, or Ier3 or Egr1 induction, by U0126 (Fig. 10).
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Functional Investigation of cAMP-Dependent, PKA-Independent Genes Regulated by PACAP as Candidate Mediators of PACAP Signaling for Neuritogenesis, Cell Size, and Growth Arrest. Based on microarray and Q-RT-PCR results, functional investigations were conducted on three transcripts (i.e., Ier3, Vil2, and Egr1) that are regulated through the cAMP/ERK pathway independently of PKA. Transfection with cognate siRNA consistently reduced Ier3, Vil2, and Egr1 induction by PACAP to the levels shown in Fig. 11A. Only Egr1 siRNA blocked PACAP-induced neurite outgrowth (Fig. 11B). This observation was confirmed by quantification of the total neurite length, which was reduced by 4-fold when cells were treated with PACAP in the presence of siRNA targeting Egr1 (Fig. 11E). Blocking Vil2 expression reduced the percentage of cells with a diameter above 17 µm, whereas Ier3 and Egr1 had no effect on cell size (Fig. 11, C-E). The effect of Vil2 on cell size was fractional (approximately 30%), suggesting that Vil2 may be only one of several effectors of altered cell size accompanying PACAP-induced PC12 cell differentiation. Neither Vil2, Ier3, nor Egr1 silencing affected growth arrest mediated by PACAP (Fig. 11, C-E), consistent with the PKA-dependent component of PACAP-induced growth arrest of PC12 cells described earlier. Among the transcripts regulated in common within the first 6 h of exposure to PACAP, dibutyryl cAMP, or forskolin, those wholly or partially dependent on PKA would be candidate mediators of growth arrest by PACAP, including both induced and pre-existing proteins regulated by NGF and reported to be involved in NGF-induced growth arrest in PC12 cells (Greene and Tischler, 1976).
Rap1 Involvement in PACAP Activation of Egr1. Silencing the expression of the cAMP- and ERK-regulated Egr1 transcript evoked the most profound and specific functional response seen in this study, inhibiting PACAP-induced neuritogenesis without affecting cell size or proliferation. We therefore focused on the activity of this trans-activator to link regulation of neuritogenesis through cAMP via ERK to PACAP-dependent activation of Rap1. An Egr1-responsive reporter gene, pEgr-Luc, driving luciferase gene expression, was transfected into PC12 cells to assay functional Egr1 activation by PACAP. PACAP treatment of transfected PC12 cells induced an approximately 10-fold increase in Egr1 reporter activity compared with vehicle (Fig. 12). To test whether Rap1 was involved in this PACAP-dependent signaling pathway to Egr1 activation, Rap1b DN was cotransfected with the Egr1 reporter before treatment with PACAP. Cotransfection of the Rap1b DN significantly reduced the PACAP-induced Egr1 reporter activity, indicating that Egr1 transcription, its functional transactivation of Egr1-dependent transcription, or both are stimulated by PACAP through Rap1 or a Rap1-like GTP-binding protein (Fig. 12).
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; Lazarovici et al., 1998), inhibition of cell division (Deutsch and Sun, 1992), stimulation of expression of neuroendocrine-specific genes such as neuropeptide Y and tyrosine hydroxylase (Corbitt et al., 1998) and changes in electrical excitability and secretion (Taupenot et al., 1999; Osipenko et al., 2000; Grumolato et al., 2003). The aim of the present study was to elucidate the signal transduction pathways leading to specific aspects of differentiation (neuritogenesis, cell size, and cell proliferation) induced by PACAP, through stringent correlation of the regulation of signaling molecules, target genes, and functional outcomes of PACAP treatment. The major finding of this investigation was that PACAP initiated a cAMP-dependent, PKA-independent activation of ERK that leads to egr1 transcription and activation, and subsequent Egr1-dependent neuritogenesis. Cyclic AMP-dependent, PKA-independent activation of ERK also leads to villin2 transcription, which is partly responsible for increased cell size after PACAP treatment. Cessation of proliferation induced by PACAP is unaffected by transcription of either of these two target genes, consistent with the partial PKA dependence of the antiproliferative effect of PACAP on PC12 cells.
We have used cDNA microarray in conjunction with biochemical analysis to establish the existence of a cAMP-dependent, PKA-independent signaling pathway responsible for activating ERK, and a discrete set of downstream target genes in PC12 cells in response to PACAP. The functional importance of this pathway was further tested by comparing the cellular and biochemical profiles observed for inhibition of PACAP- and cAMP-initiated neuritogenesis, increase in cell size, and cessation of cell division. This concordance has led in turn to the functional identification of two transcripts, those encoding Egr1 and Vil2, whose up-regulation was demonstrated through gene silencing to be required in two distinct pathways leading to PACAP-mediated neurite extension and increased cell size, respectively. Vil2 codes for a protein that has been shown to be involved in microvilli formation in intestinal epithelium by regulating actin polymerization (Craig and Powell, 1980). Some transcripts with a high sequence homology to Vil2 such as pervillin and advillin have been shown to promote neurite outgrowth in dorsal root ganglion and sympathetic neurons (Ravenall et al., 2002; Shibata et al., 2004), suggesting that a cohort of Vil2-related proteins may contribute to regulation of cell size during differentiation initiated by PACAP in PC12 cells. A third transcript, Ier3, that is prominently regulated by PACAP is apparently not involved in either of these processes and may contribute to aspects of the overall differentiation program initiated by PACAP (Taupenot et al., 1999; Osipenko et al., 2000; Grumolato et al., 2003); however, these linkages have not yet been uncovered. We have not identified any genes involved in the control of cell proliferation by PACAP. In this regard, future experiments will focus on transcripts such as Gas1, whose induction by PACAP requires PKA.
The pathways leading to cAMP-dependent and PKA-independent regulation of neurite outgrowth and cell size by PACAP are summarized in Fig. 13. These two pathways are biochemically distinct from each other, and also from the partially PKA-dependent signal transduction pathway leading to growth arrest. The combined biochemical, microarray, and gene silencing study performed here provides compelling evidence that PACAP-induced neuritogenesis proceeds through elevation of cAMP, activation of ERK through Rap1 or a Rap1-like GTP-binding protein, and subsequent increase in Egr1 trans-activation (Fig. 13).
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), and indeed phosphorylation of ERK is itself sufficient to induce neurite outgrowth (Robinson et al., 1998). However, activation of ERK per se can occur through multiple pathways with diverse functional sequelae. In fact, it has been previously reported that cAMP, PACAP, NGF, and phorbol 12-myristate 13-acetate all elicit sustained activation of ERK in PC12 cells, yet neuritogenesis stimulated by NGF, but not PACAP, proceeds through activation of Ras (Young et al., 1994; Lazarovici et al., 1998), whereas phorbol 12-myristate 13-acetate does not stimulate neuritogenesis at all. Furthermore, stimulation of ERK by NGF or cAMP leading to transin gene activation proceeds through activation of PKA (Yao et al., 1998), but that leading to neuritogenesis stimulated by PACAP does not (current study). The best working hypothesis for these disparate effects of ERK activation by distinct signaling pathways may be that both the duration of ERK signaling and the cellular compartment in which ERK activation occurs provide discrete ERK signaling "signatures" with distinct functional outcomes for the cell (MacCormick et al., 2004; Gerdin and Eiden, 2007). This hypothesis is supported by a recent report that Epac stimulates ERK through Rap1 in PC12 cells only if Epac is localized to the plasma membrane by addition of a CAAX motif and not when Rap1 is activated through endogenous Epac stimulation by 8-chlorophenylthio-2-methyl cAMP (Wang et al., 2006). It will be important to determine whether our finding of a cAMP-dependent, PKA-independent activation of ERK in the PC12 pheochromocytoma neuroendocrine cell line by PACAP corresponds to the existence of such a pathway in postmitotic primary neuroendocrine and neuronal cells. We are currently investigating this possibility, and have observed cAMP-dependent, H89-resistant ERK activation in bovine chromaffin cells (M. J. Gerdin, unpublished observations).
Egr1 is the transcript that exhibited the highest induction after NGF, PACAP, forskolin, and dbcAMP treatment, and activation of Egr1 by PACAP is dependent on the activation of Rap1. Reducing Egr1 expression with siRNA blocked the ability of PACAP to promote neuritogenesis without affecting its growth arrest effect (Fig. 13). After treatment with NGF, Egr1 acts as a transactivator to promote p35 expression, which, by binding to the cyclin-dependent kinase cdk5, induces neurite outgrowth (Harada et al., 2001). PACAP- and NGF-induced differentiation exhibit both similarities and differences in terms of mechanisms and phenotypes (Vaudry et al., 2002b). In fact, although signaling elements may be conserved in PACAP and NGF induction of neuritogenesis, our microarray analysis also shows marked differences in NGF and PACAP target gene induction. This is wholly consistent with Egr1 response element-specific transactivation by PACAP, in contrast with the intriguing results of Levkovitz and Baraban (2002) indicating that NGF stimulation of neurite outgrowth depends not on Egr1-dependent transactivation directly at genes containing an Egr1-response element but rather on Egr1 activation of c-Jun via Egr1/c-Jun heterodimerization.
Our results support the view that neurotrophin-driven differentiation is a process that occurs through simultaneous activation of multiple parallel signaling events, rather than a single common master pathway relying on a few common signaling intermediates. Neuritogenesis and cessation of proliferation initiated by NGF, for example, have been dissected into separate p53-dependent and -independent processes (Hughes et al., 2001). Likewise, it is now clear that PACAP signaling for neuritogenesis, cell size, growth arrest, and probably also for neuron-specific gene expression depend on separate, parallel pathways that diverge as early as multiple downstream targets for cAMP in PC12 cells.
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ABBREVIATIONS: NGF, nerve growth factor; dbcAMP, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate; ddAd, 2',5'-dideoxyadenosine; Egr1, early growth response 1; ERK, extracellular signal-regulated protein kinase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione transferase; H7, 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; Ier3, immediate early response 3; IRES, internal ribosome entry site; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase kinase; PAC1, PACAP specific receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; PCR, polymerase chain reaction; PD98059, 2'-amino-3'-methoxyflavone; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; Q-RT-PCR, quantitative reverse transcription-polymerase chain reaction; Rap1, member of RAS oncogene family; RBD, Rap binding domain; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; SSC, saline-sodium citrate; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio-)butadiene; Vil2, villin 2; VIP, vasoactive intestinal polypeptide; VPAC, PACAP and VIP receptor
Address correspondence to: Lee E. Eiden, Section on Molecular Neuroscience, National Institute of Mental Health, Building 49, Room 5A-68, Bethesda, MD 20892. E-mail: eidenl{at}mail.nih.gov
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