The Regulation Of C/EBPβ During Adipogenesis

Obesity is defined as an excessively high amount of body fat or adipose tissue in relation to lean body mass. The amount of body fat (or adiposity) includes concern for both the distribution of fat throughout the body and the size of the adipose tissue deposits. Both environment and genetic factors contribute to overweight and obesity.Both adipose cell hyperplasia and adipose cell hypertrophy contibute to the increasement of adipose mass .The increased number of adipocytes is from the stem cell in the adipose tissue. The stem cells commit to preadipocytes, preadipocytes can further differentiate into adipocytes.C/EBPβ plays an important role in the adipocyte differentiation program. C/EBPβ is expressed early in the program, first acting to initiate mitotic clonal expansion(MCE), and later to activate expression of C/EBPα and PPARγ, pleiotropic activators of genes that produce the adipocyte phenotype. Both MCE and expression of C/EBPα and PPARγ are required for differentiation. The transcriptional activation of the C/EBPα and PPARγ genes is mediated by the interaction of C/EBPβ with C/EBP regulatory elements in the proximal promoters of these genes. While expression of C/EBPβ occurs within 2 hours of induction of differentiation, acquisition of DNA-binding activity, and thus, transcription of the C/EBPα and PPARγ genes is delayed until much later.There are two reasons that C/EBPβ acquisition of DNA-binding activity is delay. Firstly, C/EBPβ is transiently sequestered by CHOP-10, a dominant negative regulator of C/EBPβ. The other reason is the phosphorylation modification.The expression of CHOP-10 is pretty high in growth arrested preadipocytes. It is downregulated after the treatment of differentiation cocktail, and CHOP-10 release C/EBPβ, C/EBPβ is further modified by phosphorylation.While it is still unclear why CHOP-10 is downregulated. In this paper, we did more detailed work on the regulation of CHOP-10.We also showed that C/EBPβ is sequentially phosphorylated by MAP kinase (on Thr188), followed by GSK3β(on Ser184 and Thr179) early in the differentiation program of 3T3-L1 adipocytes. We did both the in vitro phosphorylation study and in vivo functional study.Part I The regulation of CHOP-10 during the adipocytes differentiationIt is reported that the expression of CHOP-10 is increased markedly in growth arrested preadipocytes, while after induction with normal differentiation inducers, the expression of CHOP-10 is down regulated, this downregulation is concomitant with the acquisition of DNA-binding activity and localization to centromere by C/EBP β .While little is known about how CHOP-10 is downregulated during the early differentiation process. We found that fetal bovine serum (FBS) causes the down regulation of CHOP-10 during the early stage of 3T3-L1 preadipocyte differentiation and calf serum can not. High level of CHOP-10 in 3T3-L1 preadipocytes induced by calf serum with MDI cause the poor differentiation. The expression of adipocytic markers in cells induced by calf serum with MDI are also delayed and attenuated. So we can conclude that the down regulation of CHOP-10 by FBS during the early differentiation program is very important for the differentiation of 3T3-L1 adipocytes. We also found that the expression of CHOP-10 is not absolutely related with cell cycles in this paper.Part II The study of the cis-acting element and trans-acting factor that contribute to the downregulation of CHOP-10 by FBSBecause the regulation of CHOP-10 primarily happens in the transcriptional level, we construct a series of CHOP-10 promoter reporter gene recombinants to detect the possible FBS response elements. Luciferase activity analysis indicates that there is a FBS response region in CHOP-10 promoter between regions from-578bp to -321bp. Computer analysis indicates that YY1 and SRF may bind to this region and response to serum stimulation. EMSA indicates that only YY1 can bind to the region -578 to -321 of CHOP-10 promoter, and SRF cannot bind to this region. Competition EMSA confirmed that YY1 binds both the YY1 binding sites and the SRE sites which competes the binding of SRF. In our paper we also provide evidence that there are more YY1 bind to the CHOP-10 promoter in cells treated with 10% FBS than cells treated with 10% calf serum. This kind of difference may explain why CHOP-10 is downregulated by FBS during the early differentiation program.Part III Sequential phosphorylation of C/EBPβ during the adipogenesisThe expression of C/EBPβ occurs within 2 hours of induction of differentiation.Acquisition of DNA-binding activity begins after a long lag of about 14h. To elucidate the mechanism by which C/EBPβ acquires DNA binding activity, we considered the possibility that modification of C/EBPβ by phosphorylation. In the present paper we showed that C/EBPβ is sequentially phosphorylated by MAP kinase(on Thr188), followed by GSK3β(on Ser184 and Thr179) early in the differentiation program of 3T3-L1 adipocytes. In vitro phosphorylation of full-length recombinant C/EBPβ(LAP) by MAPK or GSK3β alone have no effect on the DNA binding activity of C/EBPβ. In vitro phosphorylation of full-length recombinant C/EBPβ(LAP) on Thr188 and Ser184/Thr179 by MAPK and GSK3β gives rise to DNA binding activity. The data in this study show that full-length C/EBPβ can be phosphorylated on Thr188 by MAP kinase and the phosphorylation by MAP kinase serves as the priming site for phosphorylation of Ser184/Thr179 by GSK3β. In vivo functional studies provide the further evidence that C/EBPβ is sequential phosphorylated on Thr188 by MAP kinase followed by GSK3β on Ser184/Thr179.