Abstract
The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation.
Highlights
The rat pheochromocytoma cells PC12 are a long established in vitro model to study neuronal differentiation, proliferation and survival (Greene and Tischler, 1976; Burstein et al, 1982; Cowley et al, 1994)
While AP1 was among the most persistently up-regulated transcription factors, we found a transient significance for CREB1, only, peaking at 3 and 6 h, under epidermal growth factor (EGF) or nerve growth factor (NGF) stimulation, respectively, which indicated the importance of further TFs beyond that time window
PC12 cells are a well established model to study the cellular decisions toward proliferation or differentiation
Summary
The rat pheochromocytoma cells PC12 are a long established in vitro model to study neuronal differentiation, proliferation and survival (Greene and Tischler, 1976; Burstein et al, 1982; Cowley et al, 1994). NGF binds with high affinity to the TrkA receptor (tyrosine kinase receptor A), thereby activating several downstream protein signaling pathways including primarily the protein kinase C/phospholipase C (PKC/PLC), the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and the mitogen-activated protein kinase/extracellular signalregulated kinase (MAPK/ERK) pathways (Kaplan et al, 1991; Jing et al, 1992; Vaudry et al, 2002) Beyond these immediate downstream pathways, further studies showed the involvement of Interleukin 6 (IL6), Urokinase plasminogen activator (uPA) and Tumor Necrosis Factor Receptor Superfamily Member 12A (TNFRSF12A) in PC12 cell differentiation (Marshall, 1995; Wu and Bradshaw, 1996; Leppä et al, 1998; Xing et al, 1998; FariasEisner et al, 2000, 2001; Vaudry et al, 2002; Tanabe et al, 2003). As an example for pathway crosstalk, both, the MAPK/ERK and c-Jun N-terminal kinase (JNK) pathways regulate c-Jun activity and are necessary for PC12 cell differentiation (Leppä et al, 1998; Waetzig and Herdegen, 2003; Marek et al, 2004), while uPA receptor (uPAR) signaling, as a result of transcriptional AP1 (Activator Protein-1) regulation, is necessary for differentiation of unprimed PC12 cells (Farias-Eisner et al, 2000; Mullenbrock et al, 2011)
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