| The vertebrate lens is a non-vascularized, non-innervated transparent tissue. It contains a single layer of lens epithelial cells (LECs) covering the anterior surface, while the rest is prevailingly composed of enucleated and organelle-less lens fiber cells (LFCs). The LECs are primarily responsible for maintenance of homeostasis in the ocular lens, possessing the ability of cell mitosis in a spatially defined manner throughout its life. The transformation from LECs to LFCs is a differentiation process occurred when LECs exit from their proliferation cycle. LECs differentiation process is peaked with denucleation and loss of organelles. Abnormal differentiation leads to lens pathology, i. e. cataract formation. Hitherto, numerous studies have shown that lens differentiation is regulated by an "apoptotic-like pathway". Both cell growth and differentiation in the ocular lens are regulated by different growth factors. Furthermore, the maintenance of LECs lineage and differentiation in LFCs are regulated by multiple transcription factors such as p53and c-Maf. The tumor suppressor, p53is a master regulator of apoptosis in many types of cells. Phosphorylation and dephosphorylation play a critical role in regulating p53stability and transcription factor activity. In addition to its major role in regulation of apoptosis, p53also controls cell cycle progression through its downstream gene p21. It was reported that knockout of p53causes abnormal development of mouse lens. However, the underlying molecular mechanism of how p53functions during lens development and differentiation remains largely unknown. Meanwhile, the phosphorylation status of p53during lens development and differentiation remains to be explored. In the present thesis, we address three important aspects regarding the functional mechanisms of p53in regulating lens differentiation. First of all, what is the p53expression level and phosphorylation status during lens development, especially in lens differentiation. Secondly, what proteins are associated with p53in the process of lens development and differentiation? Finally, what are the key p53downstream targets mediating the regulation of lens development and differentiation. Specifically, we conducted a series of experiments and obtained the following results.1. In this study, we principally examined the expression pattern and phosphorylation status of p53at Ser15, Ser20and Ser392during three embryonic mouse lens developmental stages (E14.5, E17.5, E19.5). Our data revealed that both p53expression level and phosphorylation level at Ser15, Ser20, Ser392, respectively were enhanced in the process of lens differentiation, as revealed by immunohistochemistry.2. We used a mouse lens epithelial cell line a TN4-1to investigate the lens differentiation in vitro. We confirmed that the formation of lentoid body in α TN4-1could mimics the process of lens differentiation in vivo. To study the function of p53, we stably knocked down endogenous p53through p53-shRNA, or inhibited the function of endogenous p53by p53-dominant negative (p53DN). As a result, we found that the rate of lentoid formation was decreased while cell proliferation and migration were not significantly affected.3. We found that high concentration of bFGF (50-100ng/ml) induced a TN4-1differentiation. An increased expression level of phosphorylated p53at Ser15, and Ser20were detected and accompanied with the increased activity of p53upstream kinases ERK1/2and Chk1. We also found an increased expression of p53downstream gene Bak, a pro-apoptotic Bcl-2family member. However, such bFGF induced α TN4-1differentiation was obviously inhibited by p53knockdown.4. The immunohistochemistry staining was conducted in p53 knockout and normal littermate mouse embryos. Our results showed that during lens development the transcription factors c-Maf, cell cycle inhibitor protein p21and lens differentiation maker a B-crystallin were downregulated.5. c-Maf expression level was upregulated, correlating with a moderate upregulation of phosphorylated p53at Ser20in the process of lentoid formation. Knocking down of endogenous p53resulted in a significant downregulation of c-Maf in α TN4-1. Thus we speculated that p53might regulate c-Maf gene transcription.6. Through bioinformatics analysis, we predicted two consensus p53binding sites in c-Maf gene. The direct binding of p53to c-Maf was confirmed by electrophoretic mobility shift assay (EMSA) in vitro. Furthermore, luciferase reporter gene assay suggested that p53significantly activated the transcriptional activity in the-584/+45region of c-Maf gene.7. Compared to the normal mouse lens, p53knockout mouse lens express less a A-crystallin. Therefore, we hypothesized that p53may transcriptionally regulated a A-crystallin expression. To test this hypothesis, we established different luciferase reporter gene constructs that contained different regions of a A-crystallin gene. The luciferase reporter gene assay suggested that p53enhanced the transcriptional activity of a A-crystallin. Additionally, we determined that p53interacted with a A-crystallin gene promoters via EMSA in vitro. Finally, the in vivo ChIP assay revealed that p53directly bind to-621/-594region of a A-crystallin, while the intron that contains p53binding sequences may act as an enhancer, which cooperate with the promoter to regulate the a A-crystallin transcriptional activity.In summary, we demonstrate that p53directly regulates c-Maf and a A-crystalline genes transcription. In addition, the spatially and temporally confined phosphorylated p53at Ser20in the process of lens differentiation suggests that this residue is likely to play a crucial role in the process of lens differentiation. Finally, our studies reveal a novel role of p53in modulating cell differentiation, and shed light on prevention and therapy of eye disease, especially cataract. |
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