Inhibition of neuronal phenotype by PTEN in PC12 cells

Abstract

The mechanisms of neuronal differentiation in PC12 cells are still not completely understood. Here, we report that the tumor suppressor PTEN has a profound effect on differentiation by affecting several pathways involved in nerve growth factor (NGF) signaling. When overexpressed in PC12 cells, PTEN (phosphatase and tensin homologue deleted on chromosome ten) blocked neurite outgrowth induced by NGF. In addition, these cells failed to demonstrate the transient mitogenic response to NGF, as well as subsequent growth arrest. Consistent with these observations was a finding that PTEN significantly inhibits NGF-mediated activation of the members of mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways, crucial for these processes. While exploring possible mechanisms of PTEN effects on NGF signaling, we discovered a significant down-regulation of both high-affinity (TrkA) and low-affinity (p75) NGF receptors in PTEN-overexpressing clones. Subsequent microarray analysis of several independent clonal isolates revealed a myriad of neuronal genes to be affected by PTEN. All of these changes were validated by quantitative PCR. Of particular interest were the genes for the key enzymes of the dopamine synthesis pathway, receptors for different neurotransmitters, and neuron-specific cytoskeleton proteins, among others. Some, but not all effects could be reproduced by pharmacological inhibitors of PI3K and/or MAPK, suggesting that PTEN may influence some genes by mechanisms independent of these signaling pathways. Our findings may shed new light on the role of this tumor suppressor during normal brain development and suggest a previously uncharacterized mechanism of PTEN action in neuron-like cells.

Fig. 1.

Effects of PTEN on NGF responses in PC12 cells. (A) Western blot analysis of PTEN levels in control (subclones C1-C4) and PTEN-overexpressing (subclones P1-P4) cells. Staining for total AKT was included as a loading control. (B) Effect of PTEN on NGF-induced neurite outgrowth in all of the isolated subclones. (C) Inhibition by PTEN of the transient mitogenic effect of NGF. (D) Effect of PTEN on NGF-induced growth arrest. For the experiments in B and C, clones C1 and P4 were selected.

Fig. 2.

Negative regulation of NGF signaling by PTEN. PC12 cells (C1 and P4) were treated with NGF, and activation of members of the PI3/AKT (A) and MEK/MAPK (B) signal transduction cascades were analyzed by Western blotting. At all time points, NGF induction of PI3K and MAPK signaling was inhibited by PTEN.

Fig. 3.

Negative regulation of NGF receptor expression by PTEN. (A) PC12 cells (C1 and P4) were treated with NGF for the indicated amount of time, and TrkA activation was analyzed by Western blotting by using antibodies specific for Tyr-674/675 and Tyr-490. Total TrkA as well as AKT (loading control) were also included. Note that reduction in phosphorylation correlated directly with overall reduction in total TrkA protein expression. (B) Suppression of TrkA and p75 mRNA levels in P4 cells as determined by Q-PCR.

Fig. 4.

Effects of pharmacological suppression of PI3/AKT and MEK/MAPK on the expression of selected genes. Control (C1) cells were treated with the indicated inhibitors separately or in combination, and the expression was analyzed by using Q-PCR 72 h posttreatment. P4 cells treated with the solvent (DMSO) were also included as a positive control. The selected genes were as follows: tyrosine hydroxylase (A), GTP cyclohydrolase I (B), p75 (C), and TrkA (D). The error bars represent the SE for two to three independent experiments for each gene.