Project description:The LIM-domain-only protein FHL2 is a modulator of signal transduction and has been shown to direct the differentiation of mesenchymal stem cells toward osteoblasts and myocytes phenotypes. We hypothesized that FHL2 may simultaneously interfere with the induction of the adipocyte lineage. Therefore, we investigated the role of FHL2 in adipocyte differentiation using pre-adipocytes isolated from mouse adipose tissue and the 3T3-L1 (pre)adipocyte cell line. Here we report that FHL2 is expressed in pre-adipocytes and for accurate adipocyte differentiation, this protein needs to be downregulated during the early stages of adipogenesis. More specifically, constitutive overexpression of FHL2 drastically inhibits adipocyte differentiation in 3T3-L1 cells, which was demonstrated by suppressed activation of the adipogenic gene expression program as shown by extensive RNAseq analyses, and diminished lipid accumulation. To identify the protein-protein interactions mediating this repressive activity of FHL2 on adipogenesis, we performed affinity-purification mass spectrometry (AP-MS). This analysis revealed the interaction of FHL2 with the Nuclear factor of activated T-cells 5 (NFAT5), an established inhibitor of adipocyte differentiation. NFAT5 knockdown rescued the inhibitory effect of FHL2 overexpression on 3T3-L1 differentiation, indicating that these proteins act cooperatively. In conclusion, we present a new regulatory function of FHL2 in early adipocyte differentiation and revealed that FHL2-mediated inhibition of pre-adipocyte differentiation is dependent on its interaction with NFAT5.
Project description:Here, we identified temporal dynamics in the proteome during adipocyte differentiation of the murine 3T3-L1 cells, applying untargeted proteomics. Samples were taken at initiation of differentiation, at an intermediate and terminal time point.
Project description:Here, we identified temporal dynamics of the phosphoproteome during adipocyte differentiation in the murine 3T3-L1 cells, applying untargeted proteomics in combination with enrichment strategies for phosphorylated peptides. Samples were taken at initiation of differentiation, at an intermediate and terminal time point.
Project description:Here, we identified temporal dynamics of the acetylome during adipocyte differentiation in the murine 3T3-L1 cells, applying untargeted proteomics in combination with immunoaffinity purification of acetylated peptides. Samples were taken at initiation of differentiation, at an intermediate and terminal time point.
Project description:Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of events including clonal expansion, growth arrest and terminal differentiation. The mechanisms coordinating these different steps are not yet fully understood. Here we investigated whether micro (mi)RNAs play a role in this process. Microarray analysis was performed to detect miRNA expression during 3T3-L1 preadipocyte differentiation. Several miRNAs, including let-7, were up-regulated during 3T3-L1 adipogenesis. Ectopic introduction of let-7 into 3T3-L1 cells inhibited clonal expansion as well as terminal differentiation. The mRNA encoding high mobility group AT-hook 2 (HMGA2), a transcription factor that regulates growth and proliferation in other contexts, was inversely correlated with let-7 levels during 3T3-L1 cell adipogenesis, and let-7 markedly reduced HMGA2 concentrations. Knockdown of HMGA2 inhibited 3T3-L1 differentiation. These results suggest that let-7 plays an important role in adipocyte differentiation and that it does so in part by targeting HMGA2, thereby regulating the transition from clonal expansion to terminal differentiation. 3T3-L1 cells were induced to differentiation into mature adipocytes using a canonical DMI cocktail. The time point at two days after confluency of 3T3-L1 was defined as day 0. Samples were collected at day 0, day 1, day 4, and day 7. The expression of microRNAs at day 1, day 4, and day 7 was compared to that of day 0.
Project description:We profiled miRNAs in 3T3-L1 adipocyte-secreted exosomes and our microarray analysis revealed that more than 300 exosomal miRNAs were detected during adipocyte differentiation.
Project description:We attempted to analyze the effect of PRMT1 knockdown in adipogenesis.3T3-L1 preadipocytes were used to investigate aipogenesis in vitro. Analysis of the transcriptomics from siNC and siPRMT1 3T3-L1 cells at MDI induction for 24 h and 72 h provides new insight into the collective roles of PRMT1 in mitotic clonal expansion and adipocyte differentiation.
