The Mechanism Of P21 Protein Expression Regulating By MAPK Signal Pathway

The cyclin-dependent kinase inhibitor p21, the product of the Cip1 gene, is critical for regulation of the cell cycle and DNA replication. It plays a role in cell differentiation, senescence, and modulation of apoptosis. p21^WAF1/Cip1 (p21) inhibits the cell cycle through its interaction with cyclin-CDK complexes. Additionally, p21 can act as an inhibitor of DNA replication through its association with PCNA, a protein required for processive DNA synthesis. Binding of p21 to PCNA affects the ability of PCNA to associate with other factors required for DNA replication.Although p21 is originally considered as a major mediator of the G1 growth arrest induced by activation of p53 in response to DNA damage, most of the studies on p21 regulation have concentrated on its transcriptional regulation by p53-independent mechanisms. A series of new studies have shown that p21 can be regulated post-translationally.p21 is a highly unstable protein with a half-life of 20-60 min in most cells. Recent studies demonstrate that proteasomal degradation of p21 is regulated by the ubiquitin pathway and suggest that the site of the ubiquitin chain is critical in making p21 a competent substrate for the proteasome.In the present study, we analyzed the regulatory effects of JNK on p21 protein accumulation in p53 null K562 cells. Results of this study showed that JNK was activated from 30 min after H₂O₂ exposure and p21 expression peaked at 4 h after cells being treated with H₂O₂. SP600125 (a JNK specific inhibitor) and Flag-JNK1 (mut) (a dominat negative construct of JNK1) decreased H₂O₂-induced p21 protein up-regulation. JNK1 also existed as a complex with p21 in K562 cells, which in accordance with previous study. Over-expression of JNK1 (WT) but not JNK1 (mut) increased endogenous p21 protein level and SP600125 not only decreased endogenous basic cellular p21 protein level but also inhibited the p21 protein accumulation induced by over-expression of JNK1 (WT). RT-PCR was performed to test the effect of JNK on p21 transcription activity. Results showed that over-expression of JNK1 (WT) did not increase p21 mRNA levels in K562 cells and H₂O₂ stimulation significantly increased p21 mRNA, but such enhancement of p21 transcription was not affected by JNK1 (mut) transfection or SP600125 treatment, suggesting that JNK post-transcriptionally mediated endogenous p21 protein accumulation. Further experiments found that over-expression of JNK1 (WT) prolonged the half-life of p21 and SP600125 reduced the half-life prolonged by H₂O₂. Finally, we test the effect of JNK on ubiquitination of p21 in vivo. Results showed that SP600125 increased the ubiquitination level of endogenous p21 and JNK decreased the ubiquitination of endogenous and transfected p21.All these findings indicated that JNK could regulate cellular p21 level via inhibiting ubiquitination of p21, which provided a new insight for analysing the regulatory effect of p21 after stress. Dipyrithione (2, 2'-dithiobispyridine-1, 1'-dioxide, PTS2), one of a pyrithione derivates, was used as bactericides, pesticides and fungicide. In this study we showed that PTS2 could induce p21 accumulation in HeLa cells, which correlated with the ERK and p38 MAPK signaling pathway activation stimulated by PTS2. Results showed that PTS2 induced p21 accumulation dramatically in HeLa cells. p21 play important roles in cell cycle arrest, so we tested cell cycle changes after exposure to PTS2 by flow cytometry. Results showed that PTS2 induced G1 cell cycle arrest in designated tumor cells. And PTS2 sinificantly decreased tumor cell number as assessed by Trypan blue compared with the control. These data consist with above p21 accumulation. We next found that, ERK and p38 activition were detected in PTS2 treated cells within a few minute. Suppression of p38 activition by SB203580 suppressed p21 induction by PTS2; however, suppression of ERK activition by PD98059 increased p21 induction by PTS2. All these data demonstrated that PTS2 can induce p21 protein level and inhibite growth of tumor cells. p21 induction by. PTS2 correlate with MAPK activation, but the detail mechanism still to be clarified.