| Adipose tissue plays an important role in energy storage. Obesity happens when caloric intake exceeds energy expenditure, leading to adipocyte hypertrophy and hyperplasia, including the recruitment of stem cells and subsequent differentiation of stromal-vascular preadipocytes. The stromal-vascular preadipocyte arises from a multi-potent stem cell population of mesodermal origin. These mesenchymal stem cells (MSCs) have the capacity to commit into several distinct cell types, including adipocytes, myoblasts, osteoblasts, and chondrocytes.A series of precise regulation happens during the process from pluripotent stem cell to mature adipocyte, including two stages:from pluripotent stem cells to preadipocyte, and from preadipocyte to terminal differentiated adipocyte. Previous studies are concentrated on the differentiation process from preadipocyte to adipocyte while knowledge about the commitment mechanism is still limited.Tang’s findings set up a good model to study the commitment process from pluripotent stem cells to preadipocyte. It has been proved that after6days treatment of10ng/ml BMP4, C3H10T1/2pluripotent stem cells can be induced into preadipocytes, and then after classical MDI cocktail treatment, they finally differentiate into mature adipocytes. The commitment process from C3H10T1/2pluripotent stem cells to preadipocyte is mainly trough BMP4and BMPR1A receptor interaction which then activates Smad and p38signal pathways.Lrpl is the member of LDL receptor family, which plays an important role in lipoprotein metabolism and degradation of proteasome. Lrpl’s fuction depends on the three important characters it owns:1, it can recognize30different ligands;2, it can interact with many cytoproteins, then transmit signal trough phosporalation;3, it can interact and regulate other membrane receptors. Tissues specific knock out of Lrpl gene reveals its key role in vascular, central nervous system and liver. Previous studies have shown that mice lacking adipocyte Lrpl (adLrp1-/-) displays reduced body weight and smaller fat stores which highlights the important role of Lrpl in adipogenesis. However, whether Lrp1influences the commitment process to change the fate of MSCs is unknown. Thus, in this paper, we use RNAi methods to knock down Lrp1in C3II10T1/2pluripotent stem cell in commitment and terminal differentiation stages respectively to test its function and find the potential mechanism. Through microscopic observation, Oil red O staining, Western blotting, it is demonstrated that Lrpl RNAi inhibits C3H10T1/2adipogenesis in commitment stage, rather than in lipid maturation period. BMP4signals through Smad1/5/8and Lrpl RNAi significantly suppresses the BMP4-triggered phosphorylation of Smad1/5/8. These data suggest that Lrpl RNAi has inhibitory effects on the BMP4-induced C3H10T1/2preadipocyte commitment via down-regulating Smad signaling. Obesity is the excess storage of body fat which leads to the increase of adipose tissue mass. The emergence of obesity depends on the increase of adipocyte number and the growth of its volume. Adipocyte, osteoblast and chondrocyte come from the same origin stem cell. Adipogenesis begins in embryonic stage and continues during the whole life span. This process is regulated by a large number of environmental factors and accompanied by the change of cell shape and its gene expression. Firstly MSCs proliferate and commits into preadipocyte, then fibrous preadipocyte differentiates into sphere adipocyte. A lot of studies demonstrate that stem cells’fate can be changed by controlling their shape and the attachment of cell and extracelluar environment.Fibronectin is the main component of extracellular matrix which interacts with the N terminal of collagen, fibrinogen and decorin. Thus fibronectin is regarded as the main regulating component of ECM biogenesis. Integrin mediates the inside-out and outside-in signal transportation, and organize cytoskeleton and transfer the signal by the interaction between different kinds of ECM.Integrin is a transmembrane protein composed by an α and a β subunit, which link cytoskeleton with ECM. Integrin a5, which belongs to integrin a family is coded by Itga5gene. After post-trascription, heavy and light chain interacts with β1subunit to compose Fibronectin receptor. Fibronectin or Itga5knocks out mice dies in embryonic period because of the development defects in mesoderm, which demonstrates the importance of Fibronectin and Itga5in mesoderm development.Itga5is of such importance to mesoderm development, while the study of adipocyte which derives from mesoderm is lack. There are even no studies of Itga5’s effect in commitment from pluripotent stem cell to preadipocyte. Take this in mind, we use C3H10T1/2pluripotent stem cell to study Itga5’s effect to commitment from pluripotent stem cell to adipocyte. C3H10T1/2cell lines come from17day year old C3H mice’s mesoderm which can commit to myocyte, bone, cartilage and adipocyte is the good model to study mesoderm development. Tang’s findings demonstrate10ng/ml BMP4promotes C3H10T1/2commits to preadipocyte, and then after the classical cocktail MDI, it differentiates into mature adipocyte. Itga5mRNA level is high in C3H10T1/2commitment stage while in differentiation stage, it is low. What’s the function of Itga5’s high expression in commitment stage. We design the Itga5RNAi, which is used to knock down Itga5in C3H10T1/2pluripotent stem cell commitment stage to resolve this question. The result demonstrates that knock down of this gene inhibits C3H10T1/2stem cell adipogenesis. We conclude that Itga5is required in C3H10T1/2pluripotent stem cell commitment. Racl is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. There are studies that Itga5can regulate active Racl expression and in3T3-L1preadipocyte, the expression profile is similar between Itag5and active Rac1. In order to find out whether Itga5play a role through Rac1in C3H10T1/2, we generate constitute active Rac1(Ca-Rac1) plasmid and overexpress it in C3H10T1/2stem cell. We find that overexpression of Ca-Racl promotes adipogenesis.Futhermore, overexpression of Ca-Rac1rescues the inhibition of adipogenesis by Itga5knockdown in C3H10T1/2pluripotent stem cells. We conclude that Rac1may be the downstream target of Itga5during the preadipocyte commitment of C3H10T1/2pluripotent stem cells. |
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