The tumor-suppressor effects of p53 are well known and are generally based upon its ability to transcriptionally activate effector proteins for cell cycle arrest, DNA damage or alternately, apoptosis. Response to DNA damage has often been performed in dividing cells. However, in differentiated, non-dividing cells that typify many tissue phenotypes in the whole organism, there is increasing evidence that p53 may affect differentiation processes, particularly in neuronal tissues. A genome-wide chromatin immunoprecipitation (ChIP) cloning technique was used in which we reported 14 new genes that are involved in making new connections between nerves (neurites) and are important in nerve cell development. Among these newly reported genes was the morphogen, wnt7b. The current project focused upon the neuritogenic activity of wnt7b in cultured cells under growth factor mediated differentiation.? ? The goals of the current research project were primarily achieved as described in a recently published report in the journal, Differentiation. The tested hypothesis was that wnt7b expression is a major soluble factor mediating NGF-dependent neurite outgrowth in differentiating neuronal cells. Wnt7b transcript and protein levels were increased after NGF via p53 shown by lowered wnt7b protein levels after stable shRNA-mediated silencing of p53. These findings are not rat specific but also appear to extend to its human transcriptional counterpart. For example, human p53 that was overexpressed in neuronal PC12 cells also produced marked wnt7b expression; however, human p53 overexpression did not elevate wnt7b levels in several tested non-neuronal human tumor cell lines suggesting the inductive effects on wnt7b might be neural-tissue specific. Ectopic wnt7b overexpression rescued neurite outgrowth in NGF-treated p53-silenced PC12 cells. Regarding the second goals, we found that neurite outgrowth could be blocked by c-Jun N-terminal kinase (JNK) inhibition with SP600125 and did not involve beta-catenin mediated nuclear translocation. Addition of soluble frizzled receptor protein, FRP1, into differentiation medium inhibited wnt7b-dependent phosphorylation of JNK, providing evidence that wnt7b is secreted and signals through a JNKdependent mechanism in PC12 cells. We further identified an NGF inducible subset of wnt receptors, Fzd 7 and 9, that likely supports wnt7b-mediated neurite extension. We project that wnt7b is a novel p53-regulated neuritogenic factor in neuronal cells and along with NGF-regulated Fzd expression is involved in p53-dependent neurite outgrowth. These effects on neurite outgrowth appear to proceed in a non-canonical fashion involving the JNK pathway. P53-controlled wnt7b expression may well be restricted to neuronal tissues under the influence of neural growth factors like NGF. ? ? Further investigation of wnt7b expression and other p53-regulated genes such as tfcp2l4/grhl3 grainyhead homolog in neuronal tissues could provide valuable knowledge on neurite outgrowth, neuronal repair processes and have implications for stem cell research in differentiation of neuronal progenitors and also in neuronal development. To investigate these possibilities, expression of the 14 genes under p53 regulation are being investigated in brain tissues of normal C57 control and p53 knockout mice. Dissected brain regions for wnt7b expression include cortex, cerebellum, hippocampus, hypothalamus, brain stem, olfactory bulb, eyes. Other non-neuronal organs involving the airway for which involvement of wnt7b in pulmonary development and disease will also be examined for comparison.
