Molecular control of adipogenesis: Mechanisms of C /EBP alpha and Wnt action.

Abstract

Obesity and its associated diseases, such as type-II diabetes and cardiovascular disease, are epidemics in the developed world today. It is estimated by the World Health Organization, that some 300 million people worldwide are clinically obese. Thus understanding basic adipocyte biology is critically important for understanding obesity as a disease. This dissertation describes experiments aimed at understanding the molecular basis of adipogenesis, the process whereby an undifferentiated cell acquires the adipocyte phenotype. The 3T3-L1 preadipocyte cell line is a well-characterized model of adipogenesis; treatment of this cell line with a cocktail of fetal bovine serum, isobutylmethylxanthine, dexamethasone and insulin (MDI) stimulates the transition from the preadipocyte to the adipocyte phenotype. Studies using these cells have revealed a cascade of transcription factors that coordinate the expression of genes critical for preadipocyte conversion to adipocytes. One of the key transcription factors in this process is CCAAT/Enhancer Binding Protein (C/EBP)alpha. Enforced expression of C/EBPalpha in 3T3-L1 preadipocytes promotes adipogenesis without MDI. Here studies are described that characterize the regions of the C/EBPalpha transactivation domain required for its ability to induce spontaneous adipogenesis. Furthermore, I show that the nuclear coactivator p300 is able to potentiate C/EBPalpha-mediated transcription through multiple regions of the C/EBPalpha transactivation domain. Stimulation of C/EBPalpha expression is mediated by the MDI-induced induction of C/EBPbeta and delta expression, in combination with the suppression of Wnt expression. To study the mechanism whereby Wnt-1 inhibits adipogenesis, microarray analyses were performed and gene expression patterns between control cells and those ectopically expressing Wnt-1 were compared. A subset of the genes involved in G₀/G₁/S-phase progression through the cell cycle are repressed in Wnt-1 cells. This prompted me to study the expression of cell cycle regulatory proteins. In this dissertation I show that the cell cycle proteins, c-Myc, E2F-4, p107, p130, p27<super>Kip1</super>, p21<super>Cip1</super>, and cyclin-dependent kinase 2, are dysregulated in cells expressing Wnt-1. This results in aberrant mitotic clonal expansion following induction with MDI. In addition to altering the expression of cell cycle genes, Wnt-1 promotes the expression of four transcriptional repressors. Experiments examining the ability of these transcriptional repressors to mimic Wnt-1 inhibition are described.