The role of C/EBPbeta in brown adipose tissue differentiation

The factors that control the conversion of non-specialised progenitor cells to differentiated cells that become tissues (e.g. adipose, muscle, liver) are a major focus of modern biology since many metabolic diseases and cancers could be cured if we understand and could control the signals responsible for differentiation in each tissue. Adipose tissue comes in two forms: white adipose tissue (WAT) which stores energy as fat, and brown adipose tissue (BAT) which oxidises fat to produce heat, due to a special gene called uncoupling protein-1 (UCP-1). The conversion of progenitor cells to WAT mainly depends on two families of gene regulators; the CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs), but we don't know very much about these factors in the conversion of progenitor cells to BAT. We know that a protein which activates members of the PPAR family called PGC-1a is highly expressed in BAT, but not in WAT, from cold stressed rodents. Furthermore, if PGC-1a synthesis is increased in progenitor cells, they start to make UCP-1 and look like brown adipose tissue cells. We have studied the control of the PGC-1a gene in brown and white cell lines and found we can switch on the PGC-1a in a white adipose tissue cell line by stimulating C/EBPbeta. We presume that the C/EBPbeta acts along with other factors related to cold stress to increase PGC-1a and convert progenitor cells into more BAT, which produces heat to help maintain body temperature. We wish to establish exactly how the C/EBPs and PPARs change during differentiation to produce BAT, and then prove that C/EBPbeta is a key regulator of BAT differentiation by increasing or decreasing its synthesis during important stages of differentiation in white and brown precursor cells. We then want to use a transgenic mouse to increase C/EBPbeta only in adipose tissue and test whether more brown adipose tissue is produced in white adipose depots during cold stress. If we are successful this will help us to increase BAT, which could stop lambs dying from cold, help treat obesity by preventing energy storage in WAT and tells us more about the biology of differentiation.

The commitment of mesenchymal stem cells to the white adipogenic lineage is dependent on a coordinated sequence of events involving two families of transcriptional factors; CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs). However, the exact sequence of expression and interaction between theses families during brown adipocyte differentiation is very poorly understood compared to white preadipocytes. The nuclear receptor coactivator PGC-1a is a 'master regulator' of brown adipose tissue differentiation since ectopic overexpression of PGC-1a is able to differentiate fibroblast and white preadipocytes cells into brown adipocytes expressing uncoupling protein-1 (UCP-1), the characteristic gene marker of brown adipocytes. We have characterised the response elements involved in cAMP induction of rodent PGC-1a gene expression in brown preadipocyte cell lines and found that a cAMP response element (CRE) in the proximal promoter is suppressed in white but not brown pre-adipocytes. However, we can activate this CRE and switch on PGC-1a expression in a white adipose tissue cell line by transient overexpression of C/EBPbeta. We hypothesise that C/EBPbeta acts directly through a cAMP response unit or indirectly (e.g. sequestering retinoblastoma protein) to increase the activation of PGC-1a gene expression by cAMP to produce a BAT specific combinatorial transcription factor control of PGC-1a expression. We wish to establish how the sequential pattern of C/EBPs and PPARs reported in white preadipocytes cell lines differs in brown preadipocytes taken from primary tissues, and then prove that C/EBPbeta is a key regulator of BAT differentiation by using tetracycline-inducible overxpression vectors in white preadipocytes or knocking down (RNAi) in brown preadipocytes. We will also employ a Tet-inducible, adipose tissue-specific transgenic mouse to establish the importance of overexpression of C/EBPbeta during cold stress in vivo.