Abstract
Neuronal differentiation of PC12 cells in response to NGF is a prototypical model in which signal duration determines a biological response. Sustained ERK activity induced by NGF, as compared with transient activity induced by EGF, is critical to the differentiation of these cells. To characterize the transcriptional program activated preferentially by NGF, we compared global gene expression profiles between cells treated with NGF and EGF for 2-4 h, when sustained ERK signaling in response to NGF is most distinct from the transient signal elicited by EGF. This analysis identified 69 genes that were preferentially up-regulated in response to NGF. As expected, up-regulation of these genes was mediated by sustained ERK signaling. In addition, they were up-regulated in response to other neuritogenic treatments (pituitary adenylate cyclase-activating polypeptide and 12-O-tetradecanoylphorbol-13-acetate plus dbcAMP) and were enriched for genes related to neuronal differentiation/function. Computational analysis and chromatin immunoprecipitation identified binding of CREB and AP-1 family members (Fos, FosB, Fra1, JunB, JunD) upstream of >30 and 50%, respectively, of the preferentially NGF-induced genes. Expression of several AP-1 family members was induced by both EGF and NGF, but their induction was more robust and sustained in response to NGF. The binding of Fos family members to their target genes was similarly sustained in response to NGF and was reduced upon MEK inhibition, suggesting that AP-1 contributes significantly to the NGF transcriptional program. Interestingly, Fra1 as well as two other NGF-induced AP-1 targets (HB-EGF and miR-21) function in positive feedback loops that may contribute to sustained AP-1 activity.
Highlights
Neuronal differentiation of PC12 cells requires sustained ERK signaling induced by NGF
Shown are the numbers of genes that were up-regulated or down-regulated at 2 or 4 h for each treatment as well as those that were preferentially affected by either growth factor. 12 rapidly induced immediate-early genes were excluded from these counts and subsequent analyses because they reached maximum induction much earlier (30 min)
The functions of the genes preferentially induced by NGF were investigated by GO analysis using the DAVID Bioinformatics Data base (30, 31). Consistent with their induction by multiple factors that induce PC12 differentiation, this gene set was enriched for GO categories related to development and differentiation, such as “developmental process” (39% of gene set), “multicellular organismal development” (32% of gene set), “system development” (29% of gene set), “tissue development” (12% of gene set), “nervous system development” (15% of gene set), and “cell differentiation” (19% of gene set) (Fig. 4A)
Summary
Neuronal differentiation of PC12 cells requires sustained ERK signaling induced by NGF. To characterize the transcriptional program activated preferentially by NGF, we compared global gene expression profiles between cells treated with NGF and EGF for 2– 4 h, when sustained ERK signaling in response to NGF is most distinct from the transient signal elicited by EGF. This analysis identified 69 genes that were preferentially up-regulated in response to NGF. The binding of Fos family members to their target genes was sustained in response to NGF and was reduced upon MEK inhibition, suggesting that AP-1 contributes significantly to the NGF transcriptional program. Fra[1] as well as two other NGF-induced AP-1 targets (HB-EGF and miR-21) function in positive feedback loops that may contribute to sustained AP-1 activity
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