The Relationship And The Molecular Mechanism Research Of P53and Mouse Preadipocyte Differentiation

Objective:The transcription factor p53is an important tumor suppressor gene; it has a fundamental role in securing the stability and integrity of the genome of normal cells. In this way, p53was suggested to be "the guardianof the genome", although its important role in growth control and neoplasia has been extensively studied, and it has been clearly described that wild type p53plays an important role in the differentiation of a variety of cell types (such as the human hematopoietic cells, hES cells and the myogenic cell line).Only a few reports have indicated that this transcription factor might play a role in adipocyte biology. Here we investigate the direct effect of p53on preadipocyte differenciation. Methods:1) Construct the plasmid of PLJM-p53-GFP;2) Establish different cell models in vitro:the overexpression group of p53in3T3-L1cells which include p53-overexpression-3T3-L1, PLJM1-3T3-L1, and3T3-L1; and the depletion group of p53in mouse embryonic fibroblasts (MEFs) which include MEF control and p53-/-MEF.3) To better understand the role of p53in preadipocytes differentiation, we firstly assayed the expression of p53protein in preadipocytes3T3-L1and MEF using western blot.4) To investigate the direct effect of p53on preadipocyte differenciation through the two cell models, firstly the cells were plated in high density (near confluence) and allowed to grow for48h before the initiation of differentiation progress, then the differentiation was stimulated with3-isobutyl-l-methyxanthine, insulin, and dexamethasone, the lipid droplets were observed by Oil-Red O staining and the triglyceride content was measured by the TG measure kit. Levels of adipogenesis-associated markers such as C/EBP-α, PPARy, LPL and AP2were measured by western blot and QRT-PCR.5) To investigate the mechanism of the effect of p53on preadipocyte differenciation, we evaluated insulin-stimulated signaling pathways, particularly the phosphorylation of AKT in MEFs. Results:1) The expression of p53increased in the fourth day of preadipocytes differentiation to adiposities, but decreased in the following eighth and tenth days.2) Through the p53overexpression in preadipocytes3T3-L1models, we observed that p53inhibited the expression of adiposities marker genes.3) While in MEFs models, the depletion of p53promote can accelerate preadipocyte MEFs differenciation, and the expression of adiposities marker genes are up-regulated.4) We evaluated insulin-stimulated phosphorylation AKT and found that decreased expression of p53can negatively influence insulin actions in MEFs. Conclusions:p53can inhibit Preadipocytes3T3-L1differentiation into adipocytes, while depletion of this gene can accelerate preadipocyte MEFs differenciation. In addition, we also discovered that p53-suppressed preadipocyte differentiation might be mediated by Akt signaling pathways.