A cAMP-Dependent, Protein Kinase A-Independent Signaling Pathway Mediating Neuritogenesis through Egr1 in PC12 Cells

Abstract

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),

Latent process genes for cell differentiation are common decoders of neurite extension length

“…S3C,G-I), the expression of the LP genes shows a distinct PKA dependency. Furthermore, addition of U0126 has been reported to inhibit PACAP-and forskolin-induced neurite extension (Hansen et al, 2003;Ravni et al, 2008), which was also confirmed in our experiment (supplementary material Fig. S5A).…”

“…In addition, we examined the effects of other members of the immediate early genes, such as Egr1 (Harada et al, 2001;Ravni et al, 2008), Fra1 and JunB (Mullenbrock et al, 2011), on NGF-induced expression of the LP genes (supplementary material Fig. S4F-K).…”

Lot1 Is a Key Element of the Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/Cyclic AMP Pathway That Negatively Regulates Neuronal Precursor Proliferation

“…We additionally found that the PAC1-R/cAMP system, up-regulated by Lot1, induced activation of the MEK pathway, with a consequent increase of c-Fos expression. According to recent studies, PACAP induces PC12 cell neurite outgrowth through either the MEK and/or the Epac pathway, independently of PKA (26,63,64). Thus, although in PC12 cells PACAP-induced neuritogenesis appears to be PKAindependent, according to our study the anti-proliferative action of PACAP is PKA-dependent.…”

The roles of protein kinases in learning and memory

“…The ERK1/2-MSK1 pathway requires cAMP production by calcium/calmodulin-dependent adenylate cyclases (Sindreu et al 2007;Xia and Storm 2012; also see Ahi et al 2004). ERK1/2 activation by cAMP involves Rap1 and is independent of PKA (Morozov et al 2003;Obara et al 2007;Ravni et al 2008). In addition to MSK1, ERK1/2 also activate ribosomal S6 protein kinase 2 (RSK2), which is mutated in Coffin-Lowry syndrome, a form of X-linked mental retardation (Schneider et al 2011).…”