Multiple signaling conduits regulate global differentiation‐specific gene expression in PC12 cells

Abstract

PC12 cells serve as a model for exploring nerve growth factor (NGF)-stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF-stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin-like growth factor-1 (IGF-1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF-1. From the set of NGF-specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra-1 and transforming growth factor beta 1 (TGF beta 1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation-dependent protein (RGT), and synapsin II required neither mitogen-activated protein kinase (MAPK) pathway. NGF-induction of the bradykinin B2 receptor and c-Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK-dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK-independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF-dependent gene expression, but additional Ras-dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation-specific gene expression in PC12 cells.

GDNF increases cell motility in human colon cancer through VEGF–VEGFR1 interaction

“…The activation of Ret by GDNF results in the activation of the MAP kinase pathways (van Weering & Bos 1997). Specifically, Ret activates the ERK and JNK signaling pathways (Marek et al 2004, Asai et al 2006. Our results showed that GDNF induced ERK, p38, JNK, and Akt phosphorylation in human colon cancer cells.…”

Temporal and functional dynamics of the transcriptome during nerve growth factor‐induced differentiation

“…In the first place, all genes that are up-or down-regulated by NGF in these studies are also up-or down-regulated in our study. Secondly, genes with a particularly strong response to NGF [Dusp6, Egr1, matrix metallopeptidase 3 (Mmp3), and Npy; FCs of at least over 10], also showed a relatively strong response in other studies that assessed expression of many genes at once (Angelastro et al 2000;Marek et al 2004;Lee et al 2005). Moreover, among these 20 genes are Dusp6, TGFb1, Mmp3, s100a4, Egr1, and Fosl1, which are reported to be regulated by NGF and not by insulin-like growth factor (Marek et al 2004).…”

“…Secondly, genes with a particularly strong response to NGF [Dusp6, Egr1, matrix metallopeptidase 3 (Mmp3), and Npy; FCs of at least over 10], also showed a relatively strong response in other studies that assessed expression of many genes at once (Angelastro et al 2000;Marek et al 2004;Lee et al 2005). Moreover, among these 20 genes are Dusp6, TGFb1, Mmp3, s100a4, Egr1, and Fosl1, which are reported to be regulated by NGF and not by insulin-like growth factor (Marek et al 2004). The latter growth factor provides trophic support to PC12 cells only, indicating that the identified genes in our study are specifically regulated during NGF-induced neuronal differentiation (Marek et al 2004).…”

“…Moreover, among these 20 genes are Dusp6, TGFb1, Mmp3, s100a4, Egr1, and Fosl1, which are reported to be regulated by NGF and not by insulin-like growth factor (Marek et al 2004). The latter growth factor provides trophic support to PC12 cells only, indicating that the identified genes in our study are specifically regulated during NGF-induced neuronal differentiation (Marek et al 2004). Up-regulation of other genes typically associated with neuronal differentiation (neuropeptide Y, b-III-tubulin, and serotonin-receptor 3a) further support the reliability of our data set.…”

“…A number of different techniques to achieve this have been employed and range from targeted display, to expressed sequence tag analysis, serial analysis of gene expression (SAGE), and microarray analysis (Lee et al 1995(Lee et al , 2005Mayumi et al 1998;Brown et al 1999;Angelastro et al 2000Angelastro et al , 2002Torocsik et al 2002;Marek et al 2004). In particular, SAGE and microarray analysis have become prominent methods to pursue these goals.…”