C/EBPβ And FoxO1Regulate Porcine Preadipocyte Differentiation Through Feedback Loop And Protein Interaction

Preadipocye differentiation is subjected to the cascade regulation of many transcriptional factors, of which CCAAT/enhancer-binding protein beta (C/EBPβ) and forkhead box O transcription factor1(FoxO1) are important members, however, the potential mechanisms of their inter-regulation in adipogenesis remain unknown. In Inflammatory, FoxO1regulates C/EBPβ expression through binding the promoter; In contrary, we analyze the transcriptional factors binding site of the FoxO1promoter and two putative sites of C/EBPβ are found. Futhermore, the domain analysis of these two proteins imply that they might form protein complex during adipogenesis. Above all, we speculate that FoxO1and C/EBPβp probably regulate adipocyte adipogenesis through transcriptional regulation mutally and protein interaction.Therefore, in our study, we used cell culture, RNAi, Oli red O staining, Real time-qPCR, WB, CoIP and dual-luciferase reporter assay to reveal their roles and mechanisms between C/EBPβ and FoxO1during adipogensis.The data are as follows:(1) FoxO1and C/EBPβ are abundant in muscle and adipose tissues of3-day-old piglets and180-day-old pigs at the mRNA and protein levels. Further, compared to the young pigs, both FoxO1and C/EBPβ mRNA and protein expressions decreased significantly in adipose tissue of180-day-old pigs (P<0.05). Additionally, during porcine preadipocyte differentiation, the level of FoxO1was lowest at day2and then increased gradually; On the contrary, C/EBPP expression increased to the peak at day2and then presented a decrease trend.(2) Three pLentiH-shRNA vectors for the target sequence of C/EBPPβ were constructed (shRNA1, shRNA2, shRNA3) and the interference efficiency of shRNA3was above70%(P<0.05). Furthermore, shRNA3treatment cells were induced adipogenic differentiation and lipid accumualation reducesd significantly, meantime, the expression of adipogenic markers (C/EBPa and aP2) exhibited a large decrease (P<0.05). Interestingly, C/EBPβ depletion decreased protein level of FoxOl markedly (P<0.05).(3) Knockdown of FoxO1incresed the lipid accumulation and western bloting data showed that the expression level of PPARy and FAS increased significantly (P<0.05). To our surprised, FoxO1interference also inhibited the protein expression of FoxO1.(4) Knockdown of both FoxO1and C/EBPβ promoted lipid accumulation largely, and increased the adipogenic markers (C/EBPa and aP2) at the pointed time of differentiation (P<0.01); In comparison with FoxO1knockdown alone, co-interference group had more significant influence on adipocyte differentiation (P<0.05).(5) The co-immunoprecipitation experiments indicated that FoxO1binded to C/EBPβ in vitro, simultaneously endogenous FoxO1also interacted with C/EBPβ in adipose tissues. Further research indicated that the interaction between FoxO1and C/EBPβ surpressed the transcriptional activity of C/EBPβ through dual-luciferase reporter assay (P>0.05), but not FoxO1(P>0.05).These results suggested that FoxO1and C/EBPβ regulate preadipocyte adipogenesis possibly through C/EBPPβ→FoxO1→C/EBPβ feedback regulatory loop and FoxO1-C/EBPPβ protein complex. This study provides a theoretical basis to improve the regulation network of adipocyte differentiation.