A cAMP-Dependent , Protein Kinase A-Independent Signaling Pathway Mediating Neuritogenesis through Egr 1 in PC 12 Cells

The neurotrophic peptide PACAP (pituitary adenylate cyclaseactivating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAP’s neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAPinduced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAP’s PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through Rap1. Neurotrophic factors activating receptor tyrosine kinases, such as nerve growth factor (NGF), promote neurite extension through a cAMP-independent signaling pathway involving Ras, PKC, and ERK (Ginty et al., 1991; 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), This work was funded by the National Institute of Mental Health (NIMH) Intramural Research Program Project 1Z01-MH002386-20, Institut National de la Santé et de la Recherche Médicale (U413), the European Institute for Peptide Research (l’Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), the Association pour la Recherche sur le Cancer and the Conseil Régional de Haute-Normandie. A.R. was the recipient of a doctoral fellowship from the Ministry of Education. Article, publication date, and citation information can be found at http://molpharm.aspetjournals. org. doi:10.1124/mol.107. 044792. ABBREVIATIONS: NGF, nerve growth factor; dbcAMP, N,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 0026-895X/08/7306-1688–1708 MOLECULAR PHARMACOLOGY Vol. 73, No. 6 U.S. Government work not protected by U.S. copyright 44792/3345219 Mol Pharmacol 73:1688–1708, 2008 Printed in U.S.A. 1688 at A PE T Jornals on Sptem er 0, 2017 m oharm . aspeurnals.org D ow nladed from 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). PACAP signaling in PC12 cells leading to neuritogenesis is distinct from that of NGF. Both processes require ERK activation, but only NGF activation of ERK is Ras-dependent (Lazarovici et al., 1998). 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 re-