Regulation Of Reactive Oxygen Species By P21

[Background]:The p21,encoded by CDKN1A,plays an important role in cell cycle regulation,cell differentiation and apoptosis.It functions via protein-protein and protein-DNA interaction.It also has a significant role in tumor development.Reactive oxygen species(ROS)are chemically active in cells.They include super oxygen anion free radical,single excited oxygen,hydroxyl free radical,superoxide and hydrogen peroxide.In recent years a large number of studies show that active oxygen is an important signal molecule,and participates in a variety of cell activities,including cell division and differentiation.However,when excessive production of ROS overwhelms the antioxidant defense system of the cells,oxidative stress ensues,which may lead to permanent cel]cycle arrest(senescence)or apoptosis.Many types of stress are inducers of ROS.Antioxidant defense systems are usually mobilized to counteract oxidative stress.There are currently two opposite views regarding the role of p21 in the regulation of ROS.There are reports that p21 possesses antioxidant effect,by its positive regulation of Nrf2.Nrf2 is usually bound and ubiquitinated by Keapl,leading to its proteosomal degradation.However,p21 can bind to Keapl competitively,thus relieving Nrf2 of the negative regulation by Keapl.Consequently,oxidative stress is decreased due to the upregulation of Nrf2.Correspondingly,depletion of p21 led to increased oxidative stress,it was also reported that p21 it can play a prooxidant effect.It was shown that depletion of p21 significantly reduced stress-induced oxidative stress.Bioinformatic analysis predicted a p53-p21-GADD45A-MAPK14-GRB2-TGF beta-mitochondrial dysfunction-DNA damage positive feedback loop that drives the elevation of ROS.We hypothesized that anti-or pro-oxidant effect of p21 might be context-and/or cell type-dependent.We therefore tested the effect of p21 in the regulation of ROS in several cell types or lines,including normal human fibroblasts(NHF),mouse skin fibroblasts(MSF),MRC5 human fibroblasts,FTE187 immortalized human fallopian tube epithelial cells.We also evaluated the effect of p21 deficiency on the proliferation and senescence in NHF and MSF.[Methods]:1.RNA interference was employed to deplete p21 in human cells.2.Cdknla knockout and wildtype mouse skin fibroblasts were prepared from knockout and wildtype mice respectively.3.DCF-based flow cytometry was used to detect the level of ROS4.Real-time PCR method was used to determine the expression of Nrf2 and its target genes NQO-1 and HO-1 in NHF and MSF cells.5.The Western blot was used to detect the changes of Nrf2 and p-p38 cells after p21 interference or knockdown.6.SA-beta gal staining technology was used to detect senescent cells.7.EdU incorporation was used to evaluate proliferation.[Results]:1.The basal as well as stress-induced level of ROS was reduced in p21 knockout MSF when compared to wildtype cells,indicating that p21 acts as a prooxidant in MSF.The difference was abrogated by the inhibition of p38,suggesting that prooxidant effect of p21 was mediated by p38 activation.Lack of p21 led to increased proliferation and decreased senescence in MSF.2.Depletion of p21 in NHF,by siRNA or shRNA,led a transient increase in ROS and a transient decrease in Nrf2.However,ROS level was reduced and NRF2 was upregulated after p21-depleted cells were grown for over a month.As in MSF,p21 depletion also led to increased proliferation and decreased senescence.3.MRC5,HET-1A and FTE187 cells responded to transient p2]depletion differently.However,the changes in NRF2 function and p38 activation were similar to those in NHF or MSF cells.[Conclusions]:The regulation of ROS by p21 is cell type and context-dependent.Lack or depletion of p21 always leads to increased proliferation and reduced cell senescence no matter transient depletion of p21 leads to a decreased or increased level of ROS.