Prevents Adipocyte Differentiation Research Articles

Specific Inhibitors of p38 Mitogen-activated Protein Kinase Block 3T3-L1 Adipogenesis

SB203580 and SB202190, pyridinyl imidazoles that selectively inhibit p38 mitogen-activated protein (MAP) kinase, are widely utilized to assess the physiological roles of p38. Here, we demonstrate that treatment of 3T3-L1 fibroblasts with these p38 MAP kinase inhibitors prevents their differentiation into adipocytes as judged by an absence of lipid accumulation, a lack of expression of adipocyte-specific genes, and a fibroblastic morphological appearance. In 3T3-L1 fibroblasts and developing adipocytes, p38 is active. p38 activity decreases dramatically during later stages of differentiation. In accordance with the time course of p38 activity, p38 inhibitor treatment during only the early stages of differentiation is sufficient to block adipogenesis. In addition, we constructed a 3T3-L1 cell line harboring an inducible dominant negative p38 mutant. The induction of this dominant negative mutant of p38 prevents adipocyte differentiation. Thus, it is likely that the antiadipogenic activity of SB203580 and SB202190 is indeed due to inhibition of p38 MAP kinase. This study points out that CCAAT/enhancer-binding protein beta (C/EBPbeta), a transcription factor critical for the initial stages of 3T3-L1 adipogenesis, bears a consensus site for p38 phosphorylation and serves as a substrate for p38 MAP kinase in vitro. Although the induction of C/EBPbeta is not significantly altered in the presence of the p38 inhibitor, the amount of in vivo phosphorylated C/EBPbeta is reduced by SB203580. The transcriptional induction of PPARgamma, a gene whose expression is induced by C/EBPbeta, and a factor critically involved in terminal differentiation of adipocytes, is impaired in the presence of p38 inhibitors. Thus, transcription factors such as C/EBPbeta that promote adipocyte differentiation may be p38 targets during adipogenesis. Collectively, the data in this study suggest that p38 MAP kinase activity is important for proper 3T3-L1 differentiation.

Human Translocation Liposarcoma-CCAAT/Enhancer Binding Protein (C/EBP) Homologous Protein (TLS-CHOP) Oncoprotein Prevents Adipocyte Differentiation by Directly Interfering with C/EBPβ Function

Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBPbeta in naïve NIH3T3 cells, and their ability to inhibit C/EBPbeta-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBPbeta and inhibit C/EBPbeta DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP's putative transactivation activity. Another adipogenic transcription factor, PPARgamma2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBPbeta from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process.

Notch-1 Controls the Expression of Fatty Acid-activated Transcription Factors and Is Required for Adipogenesis

Notch, a transmembrane receptor member of the homeotic epidermal growth factor-like family of proteins, participates in cell-to-cell signaling to control cell fate during development. Activated Notch-1 constructs lacking the extracellular region prevent differentiation of several mammalian cells in vitro. This effect, however, bypasses the normal mechanisms of cell-to-cell interactions in which Notch-1 participates. We investigated the role of Notch-1 in the hormone-induced adipocyte differentiation of 3T3-L1 fibroblasts, a paradigmatic model of adipogenesis that requires cell-to-cell contact. Unlike other differentiation models, Notch-1 expression and function were necessary conditions for adipogenesis. Impaired Notch-1 expression by antisense Notch-1 constructs prevented adipocyte differentiation. Strategies aimed at blocking putative Notch/ligand interactions also blocked adipogenesis, implicating Notch as a critical molecule in cell-to-cell signaling necessary for differentiation. Inhibition of Notch-1 expression or function decreased the expression of peroxisomal proliferator-activated receptors delta and gamma, transcription factors that control adipocyte differentiation and that are up-regulated at cell confluence. These results implicate Notch in the commitment of 3T3-L1 cells to undergo adipogenesis by controlling the expression of the principal regulators of this process.