Nerve growth factor regulation of transcription factorp53 activity.

摘要

p53 is recognized as a critical regulator of the cell cycle and apoptosis. Mounting evidence also suggests a role for p53 in the differentiation of several cell types including neuronal precursors. Using the PC12 cell model derived from adrenal chromaffin cells of ectodermal lineage, we studied the transcriptional role of p53 during neuronal differentiation induced by nerve growth factor (NGF) treatment. Differentiation in PC12 cells by NGF is characterized by growth arrest and neurite extension. The transcription factor p53 contributes to each of these processes but the mechanisms are incompletely understood. We hypothesized that p53 contributed to PC12 differentiation through the regulation of gene targets distinct from its known transcriptional targets during apoptosis. This study demonstrated that p53 protein was transcriptionally activated and contributed to NGF-mediated neurite outgrowth during differentiation of PC12 cells. Using a genome-wide chromatin immunoprecipitation cloning technique, we identified and validated 14 novel p53-regulated genes following NGF treatment. Furthermore, we describe stimulus-specific regulation of a subset of these target genes by p53. The most salient differentiation-relevant target genes included wnt7b involved in dendritic extension and the tfcp2l4/grhl3 grainyhead homolog implicated in ectodermal development. Additional targets included brk, sdk2, sesn3, txnl2, dusp5, pon3, lect1, pkcbpb15 and other genes. These studies demonstrate that receptor-mediated p53 transcriptional activity is involved in PC12 differentiation and may suggest a contributory role for p53 in neuronal development.; Since NGF signaling stabilizes p53 and enables the transcriptional regulation of various target genes, including wnt7b, we tested the hypothesis that wnt7b expression is involved in p53-mediated neurite outgrowth in NGF-differentiated PC12 cells. Wnt7b transcript increased within 3 days, while protein levels of wnt7b are rapidly and efficiently increased within 24 hours of NGF exposure. Stable silencing of p53 by shRNA reduced wnt7b protein levels and halted neurite outgrowth in NGF-treated cells. Immunofluorescence showed wnt7b was distributed throughout the cytosol but changed to peri-cytoplasmic, nodal localization during wnt7b overexpression in transfections of mitotic and NGF-differentiated cells. Overexpressed wnt7b produced marked neurite extensions in the presence of NGF and was sufficient to restore neurite outgrowth in p53-silenced cells. Therefore, wnt7b is a p53-regulated neuritogenic factor that in conjunction with NGF signaling is capable of eliciting potent induction of neurite outgrowth in PC12 cells.; NGF is also recognized for its role in neuronal differentiation and maintenance. We investigated NGF influence over p53 activity during NO-induced apoptosis by sodium nitroprusside in differentiated and mitotic PC12 cells. NGF-differentiation produced increased p53 levels, nuclear localization and sequence-specific DNA binding. Apoptosis in mitotic cells also produced these events but the accompanying activation of caspases 1-10 and mitochondrial depolarization were inhibited during NGF differentiation and could be reversed in p53-silenced cells. Transcriptional regulation of PUMA and survivin expression were not inhibited by NGF, although NO-induced mitochondrial depolarization was dependent upon de novo gene transcription and only occurred in mitotic cells. Therefore, NGF mediates prosurvival signaling through factors such as Bcl-2 and p21Waf1/Cip1 without altering p53 transcriptional activity to inhibit apoptosis.; These studies demonstrate that NGF potently activates p53 transcriptional regulation of target genes involved in cell cycle arrest and neurite outgrowth. Although NGF potentiates p53 DNA-binding activity, it also inhibits p53-dependent apoptosis. In summary, receptor-mediated NGF signaling represents a comprehensive mechanism through which many functions of p53 activity ma