Identification of new Nerve Growth Factor-responsive immediate-early genes.
Stimulation of the PC12 pheochromocytoma cell line with the prototypical neurotrophin Nerve Growth Factor (NGF) induces a cellular response of neuronal differentiation and is therefore a widely used model to gain molecular insight into this process. Classically, the transcriptional response to extracellular stimuli such as NGF is divided in genes that require no protein synthesis prior to their induction (immediate-early genes) and genes that do (delayed-response genes). Because an increasing number of studies have reported important roles for immediate-early genes (IEGs) in neuronal differentiation, the goal of the present study was to identify previously unrecognized NGF-responsive IEGs. Stimulation with NGF for 15, 30, 60 and 120 min resulted in a typical transient induction of many known NGF-responsive IEGs. To identify candidate new genes, we analyzed 27000 measured expression profiles and selected 10 genes for further study. Five genes, including Cbp/p300-interacting transactivator 2 (Cited2), Kruppel-like factor 4 (Klf4), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein F (Maff), Kruppel-like factor 10 (Klf10 or Tieg) and Activating transcription factor 3 (Atf3) were selected and positively validated by qPCR. NGF-induced activation of all five genes seems to be mediated by MAPK and PI3K-mediated pathways. Additionally, we tested translation-independent induction and showed that NGF induced upregulation of these genes in both the subclonal Neuroscreen-1 PC12 and parental PC12 cell line. These 5 transcription factors have not been previously reported as NGF-responsive IEGs, however have previously been reported as important regulators of cell differentiation and proliferation in different systems. These observations may therefore provide important new information on the molecular mechanisms underlying NGF-induced differentiation.
Dijkmans
T F
TF
Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden University Medical Center, PO Box 9502, 2300 RA, Leiden, The Netherlands. tdijkmans@gmail.com
van Hooijdonk
L W A
LW
Schouten
T G
TG
Kamphorst
J T
JT
Fitzsimons
C P
CP
Vreugdenhil
E
E
D051700:Activating Transcription Factor 3 / Q000096:biosynthesis / Q000235:genetics; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D017781:Genes, Immediate-Early / Q000502:physiology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D046228:Microarray Analysis; D020932:Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D051256:Oncogene Protein v-maf / Q000235:genetics; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000235:genetics
D051700:Activating Transcription Factor 3; C497108:Atf3 protein, rat; C517260:CITED2 protein, rat; D004268:DNA-Binding Proteins; C491385:Klf10 protein, rat; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D051256:Oncogene Protein v-maf; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D014157:Transcription Factors; D020932:Nerve Growth Factor