| Cell apoptosis plays an important role in tumorgenesis, embryonic development, immune response, nervous system development, as well as metabolism of tissues and cells. Abnormal apoptosis is one of the key mechanisms for many severe diseases threatening the health of human beings. For example, abnormal apoptosis is critical in the pathogenesis and progress of many tumors. However, despite of numerous studies being performed on the regulation of apoptosis, there still exist various conflicts on the phenomenon of cell apoptosis, which significantly affects the basic research and clinical therapy of tumor. Therefore, it is necessary to clarify these conflicting results for the breakthroughs in the basic research field of tumor and for the improvement of clinical treatment of tumor.Mitogen-activated protein kinases (MAPK) are important signal molecules transducing extracellular signals from the membrane to the nuclei of the cells, p38 MAP kinase, representing one of the four MAP kinase subfamilies in the mammalian cells, is mainly involved in the regulation of stress and inflammation. Further studies show that p38 MAPK also plays a role in the regulation of cell transformation and tumor metastasis, and the regulation on cell cycle and apoptosis is the important cellular function for this kinase. The biological effects mediated by the activation of p38 MAPK may be distinct and even opposite in different cells treated with stimulations of various intensities.In addition, the results may be significantly different based on the methods employed in the experiments. For example, serveral previous studies showed that p38 MAPK was phosphorylated and activated to mediate apoptosis of the cells treated with various stimuli. On the contrary, there were also some reports indicating that p38 MAP kinase has protective effects on the cells under certain circumstances. Therefore, there still exist arguments in the biological effects of p38 MAPK activation on the regulation of apoptosis.p53 is one of the most intensively studied tumor suppressor gene at present. Wild type p53 is involved in many pathophysiological processes including DNA repair, cell cycle regulation, apoptosis and angiogenesis inhibition, etc. The mutations of p53 gene bring about lose of functions described above, leading to the formation of tumor. Although it is well accepted that activation of p53 which functions as a 'molecular policeman' bring about the the effects of cell cycle arrest and apoptosis. However, there are also some studies indicate that the activation of p53 under certain circumstances lead to cell survival. This suggests that the activation of p53 pathway not only leads to growth inhibition and apoptosis, but also activates the survival-related pathway on the harmful stimulations, such as DNA damage, to achieve a balance between the cell survival and cell death. Therefore, it is reasonable to predict that the ultimately fate of the cells, apoptosis or survival, dependent on the type and intensity of stimulation, as well as the cell types used.Many studies showed that there are important functional links between p38 MAPK and p53 on the regulation of cell apoptosis. For example, ultraviolet radiation B (UVB) radiation leads to the activation of p38 MAP kinase in normal keratinocytes,and then the activated p38 MAP kinase phosphorylates p53 on Ser15, resulting in the inhibition of nuclear translocation of p53. By this way, p38 and p53 protect the cells from UV radiation. Yet in another example, nitric oxide (NO) induced phosphorylation of p38 in rabbit articular cartilage, and tne activated p38 phosphorylates p53 on Ser15 leading to cell apoptosis. These results suggest that the phosphorylation and activation of p53 by p38 MAPK may lead to either cell apoptosis or cell survival. On the one hand p53 is activated by p38 MAPK, on the other hand p53 has effects on p38 MAPK through the regulation on transcriptional activity of the promoter. For example, a protein Wipl regulates p38-p53 pathway through a feedback mechanism, to prevent this pathway from excessive activation.Although the opinion that p38 MAPK and p53 both have very important roles in the regulation of cell apoptosis has been widely accepted, some confilicta still exist on the exact mechanisms of the both, due to the variations of experimental methods, thchnique systems and cell lines employed in different cases.As a pivotal method in the research of protein functions, the results from gene knockout animals are more reliable than other methods, and it is conclusive in the demonstration of protein functions with gene knockout experiments. Hence, it is necessary to employ the p38 MAPK or p53 gene knockout cell line to study their exact physiological functions under different stimulations, in order to clarify the functions of p38 and p53 and their functional links in the regulation of cell apoptosis.In this study, we selected UV radiation as the stimulation. Firstly, we studied the dunamic process of p38 MAP kinase activation upon UV radiation. The results showed that p38 MAPK was notably activated 30 min after UV radiation, reached the peak of activation within 60 to 120 min approximately, and finally came back to the basal level within 240 min. Different from p38 MAP kinase, p53 was phosphorylated 15 min after UV radiation, reached the peak of activation within 60 min, and keep the high level of phosphorylation continuously. These results suggested that the dynamic process of UV-induced phosphorylations of p38 MAPK and p53 were different.To investigate if the phosphorylation of p53 was mediated by p38 MAP kinase under the stimulation of UV radiation, a p38 MAP kinase specific inhibitor, SB203580, was employed to study the effect of p38 MAP kinase inhibition on p53 phosphorylation. The results showed that p38 MAP kinase and its downstream substrate ATF-2 were phosphorylated, and SB203580 pretreatment had no effect on its own phosphorylation, but inhibited the kinase activity of p38 MAP kinase. Meanwhile, we also found that p53 was phosphorylated and activated, and SB203580 pretreatment had no blocking effect on the activation of p53, suggesting that the phosphorylation of p53 is not mediated by p38 MAP kinase and the both proteins mediate UV-induced cell responses through different mechanisms.Having verified the expression of p38 MAP kinase was defective in the p38 gene knockout cell line by Western blot and immunostaining, we further explored the effects of p38 gene knockout on UV-induced phosphorylation of p53. The results showed that under the stimulation of UV radiation, p53 was notably activated in both p38+/+ and p38-/- cells, suggesting that p38 MAP kinase gene knockout has no blocking effect on UV-induced phosphorylation of p53, and p38 MAPK is not the major kinase for the phosphorylation on Ser15 of p53 under the stimulation of UV radiation.We further studied if the UV-induced nuclear translocation of p53 was blocked in p38 MAP kinase gene knockout cell line. The results showed that the intracellular localization of p53 in resting p38-/- cells was similar to that of p38+/+ cells, and under the stimulation of UV radiation, the activation of p53 was not blocked as well as its nuclear translocation.Having verified that the expression of p53 was defective in the p53 gene knockout cell line by Western blot and immunostaining, we further studied the effects of p53 gene knockout on UV-induced p38 MAP kinase phosphorylation. The results showed that under the stimulation of UV radiation, p38 MAP kinase were notably activated in both p53+/+ and p53-/- cells, suggesting that p53 gene knockout has no blocking effect on UV-induced phosphorylation of p38 MAP kinase.Then, we studied if the nuclear translocation of p38 MAP kinase was blocked in p53 gene knockout cells stimulated by ultraviolet. The results showed that the intracellular localization of p38 MAP kinase in the resting p53-/- cells was similar to that of p53+/+ cells, and under the stimulation of UV radiation, both the activation of p38 MAP kinase and its nuclear translocation was not blocked in p53-/- cells. Having certified that interference to p38 MAPK or p53 had no effect on the phosphorylation and nuclear translocation of the other, we tested if there was a physical interaction between them by co-immunoprecipitation experiment. The results showed that there was no interaction between them under the stimulation of UV radiation.We then studied the effects of p53 or p38 MAPK gene knockout on UV-induced cell apoptosis with fluorescence microscopy. The results showed that the nucleus staining of p38+/+ cells were uniform, while a small fraction of p38-/- cells displayed nuclear condensation even in resting cells; after the stimulation of UV radiation, there were no visible changes in p38+/+ cells, while a portion of p38-/- cells showed some typical apoptotic changes, such as nuclear condensation, chromatin margination and nuclear fragmentation. In resting cells, the nucleus staining of p53+/+ and p53-/- cells were both uniform. After UV radiation, there were no visible changes in p53+/+ cells, while a portion of p53-/- cells showed typical changes of apoptotic cells. These results suggested that both p38 MAP kinase and p53 had protective effects on UV-induced cell apoptosis.After having studied the effects of p53 or p38 MAPK gene knockout on UV-induced cell apoptosis with morphological methods, we next performed quantitative analysis of cell apoptosis with flow cytometry. We first studied the effect of p38 MAPK gene knockout. The results showed that the ratio of apoptotic p38-/- cells induced by UV significantly increased compared to that of the untreated cells (P＜0.01), and also significantly increased compared to that of p38+/+ cells stimulated with UV (P＜0.01). We then studied the effect of p53 gene knockout on UV-induced cell apoptosis. The results showed that the apoptotic ratio of p53-/- cells treated with UV significantly increased compared to that of the untreated cells (P＜0.01), and also significantly increased compared to that of p53+/+ cells induced by UV (P＜0.01).These results suggested that p38 MAP kinase or p53 gene knockout really made the cells prone to apoptosis under the stimulation of UV radiation. Next, we observed the effects of SB203580 pretreatment on UV-induced apoptosis of p53 or p38 MAPK gene knockout cells. The results showed that pretreated with SB203580, the apoptotic cell ratios of p38+/+ or p38-/- cells induced by UV significantly increased compared to that of the untreated cells, respectively (P＜0.01), but there was no difference between the p38+/+ and p38-/- cells treated with UV. Meanwhile, the apoptotic ratios of p53+/+ or p53-/- cells induced by UV significantly increased compared to that of the untreated cells, respectively (p53+/+ P＜0.05; p53-/- P＜0.01). In addition, pretreatment with SB203580 alone led to an increase on the apoptotic ratios of p53-/- cells compared to that of p53+/+ cells.(P＜0.05). Consistantly, SB203580 pretreatment followed by UV radiation displayed a similar result in p53-/- cells and p53+/+ cells (P＜0.01). These results suggested that not only one of p38 MAP kinase and p53 gene knockout made the cells prone to apoptosis under the stimulation of UV radiation, but also these two proteins had additive effects of cell protection. Although both p38 MAP kinase and p53 had protective effects on the cell apoptosis induced by UV radiation, there were no functional links between these two proteins on the activation dynamic, enzyme activation by each other, nuclear translocation and physical interactions, suggesting that they mediate the function of cell protections via different cellular signaling mechanisms.Taken together, in this paper we studied the activation, nuclear translocation, and interaction of p38 MAPK and p53 and apoptosis in the cells using immunoblotting, immunostaining, nuclear morphology observation and flow cytometry to explore the probable mechanisms of cell protections of these two proteins under low intensity UV radiation. Several conclusions drawn as follows:1. There are different characteristics of the dynamic activation of p38 MAP kinase and p53 upon UV radiation.2. There are no effects of p38 MAPK inhibition or deficiency on UV-induced p53 phosphorylation and nuclear translocation.3. There are no effects of p53 deficiency on UV-induced p38 phosphorylation and nuclear translocation;4. Both p38 MAPK and p53 have protective effects on the low intensity UV radiation-induced cell apoptosis and they are functionally additive;5. The cell protection effects of p38 MAPK and p53 on UV radiation-induced cell apoptosis were achieved through different molecular mechanisms.
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