Study On Roles Of ERK Pathway, P53/P21 And CDK5/P35 During NGF-induced Differentiation Of PC12 Cells

Objective: To investigate the roles and relationships among ERK pathway, P53/P21 and CDK5/P35 during neuronal differentiation using a differentiative model of PC12 cells induced by NGF.Methods: A new cell line PC12(P53/ml75) was created by stable transfection of a retrovirus plasmid pBabe-P53/m175 , which contains a dominant-negative p53 gene mutant. After NGF treatment, western blotting of P53 and P21, flow cytometric analysis and growth curve assay were performed to compare with the control group. The protein expression of P35 and CDK5 was assayed in normal PC 12 cells group and a CDK5 inhibitor, roscovitine-preincubated group after NGF treatment. Phase-contrast microscopy was used to observe morphological characteristics and neurite outgrowth of different groups. Using the specific inhibitor U0126 of ERK pathway and co-transfection of pCMV-CDK5, pCMV-P35 to PC12 cells, the protein levels of P53, P21, P35 and CDK5 were analyzed by Western blotting, and the proccesses of neurite outgrowth were observed using phase-contrast microscopy to get the differences between experimental groups and control groups.Results: Expression of P53 and P21 was obviously increased in NGF-induced PC 12 cells, which began at about 12h after NGF treatment, reached a plateau at 40h and lasted at least 4 days. The appearance of cell cycle G1 phase arrest paralleled the increased expressions of P53 protein and P21 protein. The level of P21 protein did not change after treatment with NGF in PC12(P53/ml75) cells and the extent of G1 phase arrest markedly decreased by contrast with the control group. However, we could observe the normal neurite outgrowth in NGF-treated PC12(P53/ml75) cells. The amount of P35 protein increased strongly after NGF treatment, while the protein levels of CDK5 were not change obviously. Once a CDK5 inhibitor, roscovitine was used, NGF-induced neutite outgrowth was severely impaired, and morphological changeswere no longer observed. The activation of ERK pathway in the early stage was observed in both PC12(P53/ml75) cells and roscovitine-incubated PC12 cells after NGF treatment. We used a specific inhibitor of ERK pathway, U0126 to completely inhibit activation of ERK, which led to the significant blockage of the increased expression of P53, P21 and P35 and the inactivation of CDK5. The differentiating phenotyes of U0126-treated PC12 cells were also inhibited to a high degree. If these cells were co-trasfected with pCMV-CDK5, pCMV-P35, inhibition of NGF-induced neurite outgrowth by U0126 was partially rescued by overexpression of CDK5 and P35. But the expression of CDK5 and P35 was not sufficient to fully overcome the inhibition.Conclusions: The activation of ERK pathway in the early stage after NGF treatment is necessary for neuron-like differentiation of PC12 cells. NGF-induced the increased protein levels of P53 and its transcriptional element, P21 is essential for cell cycle G₁ phase arrest, but does not necessarily exclusively correlate with the neurite outgrowth of PC12 cells. The sustained and strong expression of P35 and thus prolonged activation of the kinase activity of CDK5 play a crucial role in NGF-induced neurite outgrowth in PC12 cells after NGF treatment. Both P53/P21 and CDK5/P35 are located in the downstream of ERK pathway, and their activations induced by NGF rely on a mechanism that ERK pathway needs to activated at first.