Mice deficient in C/EBP alpha have defective development of adipose tissue, but the precise role of C/EBP alpha has not been defined. Fibroblasts from C/EBP alpha(-/-) mice undergo adipose differentiation through expression and activation of PPAR gamma, though several clear defects are apparent. C/EBP alpha-deficient adipocytes accumulates less lipid, and they do not induce endogenous PPAR gamma, indicating that cross-regulation between C/EBP alpha and PPAR gamma is important in maintaining the differentiated state. The cells also show a complete absence of insulin-stimulated glucose transport, secondary to reduced gene expression and tyrosine phosphorylation for the insulin receptor and IRS-1. These results define multiple roles for C/EBP alpha in adipogenesis and show that cross-regulation between PPAR gamma and C/EBP alpha is a key component of the transcriptional control of this cell lineage.

The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) ␥ is a ligand-activated transcription factor that regulates several crucial biological processes such as adipogenesis, glucose homeostasis, and cell growth. It is also the functional receptor for a new class of insulin-sensitizing drugs, the thiazolidinediones, now widely used in the treatment of type 2 diabetes mellitus. Here we report that PPAR␥ protein levels are significantly reduced in adipose cells and fibroblasts in response to specific ligands such as thiazolidinediones. Studies with several doses of different ligands illustrate that degradation of PPAR␥ correlates well with the ability of ligands to activate this receptor. However, analyses of PPAR␥ mutants show that, although degradation does not strictly depend on the transcriptional activity of the receptor, it is dependent upon the ligand-gated activation function 2 (AF2) domain. Proteasome inhibitors inhibited the down-regulation of PPAR␥ and ligand activation enhanced the ubiquitination of this receptor. These data indicate that, although ligand binding and activation of the AF2 domain increase the transcriptional function of PPAR␥, these same processes also induce ubiquitination and subsequent degradation of this receptor by the proteasome.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer that frequently harbours Merkel cell polyomavirus (MCV) DNA integrated in the genome of the tumor cells. In our study, we elaborate our recent finding that MCV-positive MCC cell lines require the expression of the viral T antigens (TA). Indeed, in a xeno-transplantation model, we prove that TA expression is essential also in an in vivo situation, as knock down of TA leads to tumor regression. Moreover, rescuing TA short hairpin RNA (shRNA)-treated MCV-positive MCC cells by ectopic expression of shRNA-insensitive TAs clearly demonstrates that the observed effect is caused by TA knockdown. Notably, introduction of a mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) ablated this rescue. The importance of this interaction was further confirmed as LTA-specific knockdown leads to explicit cell growth inhibition. In summary, the presented data demonstrate that established MCV-positive MCC tumors critically depend on TA expression, in particular the LTA and RB interaction, for sustained tumor growth. Consequently, interference with LTA/RB interaction appears as promising strategy to treat MCC.

While searching for natural ligands for the peroxisome proliferator-activated receptor (PPAR) ␥, we identified a synthetic compound that binds to this receptor. Bisphenol A diglycidyl ether (BADGE) is a ligand for PPAR␥ with a K d(app) of 100 M. This compound has no apparent ability to activate the transcriptional activity of PPAR␥; however, BADGE can antagonize the ability of agonist ligands such as rosiglitazone to activate the transcriptional and adipogenic action of this receptor. BADGE also specifically blocks the ability of natural adipogenic cell lines such as 3T3-L1 and 3T3-F442A cells to undergo hormone-mediated cell differentiation. These results provide the first pharmacological evidence that PPAR␥ activity is required for the hormonally induced differentiation of adipogenic cells. Peroxisome proliferator-activated receptor (PPAR)1 ␥ is a nuclear hormone receptor that is expressed at highest levels in adipose tissue and lower levels in several other tissues. PPAR␥ is a major coordinator of adipocyte gene expression and differentiation (1). The expression of this receptor occurs early during the differentiation of preadipocytes, and it is expressed in a highly adipose-selective manner.PPAR␥ has been considered an orphan member of the nuclear hormone receptor superfamily, because no high affinity endogenous ligand has been identified for this receptor. However, a number of synthetic compounds have been shown to bind and activate PPAR␥ including a relatively new class of antidiabetic drugs, the thiazolidinediones (2). Thiazolidinediones (TZD) can ameliorate glucose metabolism and improve whole body insulin sensitivity in many animal models of obesity and diabetes. One TZD, troglitazone (Rezulin TM ), is currently used in the treatment of Type II diabetes in humans, and a second, rosiglitazone (Avandia TM ), was recently approved by the United States Food and Drug Administration. In addition to synthetic ligands, a number of natural ligands have been described for PPAR␥ that include primarily fatty acids and their metabolites (3-5). These ligands, however, have relatively low affinities with K d Ϸ 2-50 M, and hence it is possible that, analogous to other nuclear hormone receptors, a higher affinity ligand for PPAR␥ might exist.The evidence supporting a key role for PPAR␥ in adipogenesis is strong, but it is entirely based on "gain of function" experiments. For example, it has been shown that the ectopic expression and activation of PPAR␥ in undetermined fibroblasts are sufficient to induce an adipogenic response that includes morphological changes, lipid accumulation, and expression of most of the genes characteristic of this cell type (6). However, until now no experiments have addressed whether PPAR␥ function is required for adipocyte differentiation. During a screen for endogenous ligands of PPAR␥ we purified and characterized a compound that exhibited PPAR␥ binding activity. High pressure liquid and gas chromatography/mass spectrometry (LC/MS/MS and GC/MS, respectively)-based analyses identified this ...