Summary Lineage conversion of differentiated cells in response to hormonal feedback has yet to be described. To investigate this we studied the adrenal cortex, which is composed of functionally distinct concentric layers that develop postnatally, the outer zona Glomerulosa (zG) and the inner zona Fasiculata (zF). These layers have separate functions, are continuously renewed in response to physiological demands and are regulated by discrete hormonal feedback loops. Their cellular origin, lineage relationship and renewal mechanism, however, remain poorly understood. Cell fate mapping and gene deletion studies using zG-specific Cre expression demonstrate that differentiated zG cells undergo lineage conversion into zF cells. In addition, zG maintenance is dependent on the master transcriptional regulator Steroidogenic Factor 1 (SF-1) and zG-specific Sf-1 deletion prevents lineage conversion. These findings demonstrate that adrenocortical zonation and regeneration result from lineage conversion and may provide a paradigm for homeostatic cellular renewal in other tissues.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrate that human breast cancer cells, but not normal mammary epithelial cells, are dramatically sensitized to TRAILinduced apoptosis and caspase activation by peroxisome proliferator-activated receptor ␥ (PPAR␥) agonists of the thiazolidinedione (TZD) class. Although TZDs do not significantly alter the expression of components of the TRAIL signaling pathway, they profoundly reduce protein levels of cyclin D3, but not other D-type cyclins, by decreasing cyclin D3 mRNA levels and by inducing its proteasomal degradation. Importantly, both TRAIL sensitization and reduction in cyclin D3 protein levels induced by TZDs are likely PPAR␥-independent because a dominant negative mutant of PPAR␥ did not antagonize these effects of TZDs, nor were they affected by the expression levels of PPAR␥. TZDs also inhibit G 1 to S cell cycle progression. Furthermore, silencing cyclin D3 by RNA interference inhibits S phase entry and sensitizes breast cancer cells to TRAIL, indicating a key role for cyclin D3 repression in these events. G 1 cell cycle arrest sensitizes breast cancer cells to TRAIL at least in part by reducing levels of the antiapoptotic protein survivin: ectopic expression of survivin partially suppresses apoptosis induced by TRAIL and TZDs. We also demonstrate for the first time that TZDs promote TRAIL-induced apoptosis of breast cancer in vivo, suggesting that this combination may be an effective therapy for cancer.

Peroxisome proliferator-activated receptor-␥ (PPAR␥), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPAR␥ mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPAR␥ L466A mutation, shown previously to inhibit wild-type PPAR␥ action in vitro. Homozygous PPAR␥ L466A knock-in mice die in utero. Heterozygous PPAR␥ L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPAR␥ function and the metabolic syndrome.The metabolic syndrome, or syndrome X, is characterized by a constellation of insulin resistance, dyslipidemia, obesity, and hypertension (1). The prevalence of the metabolic syndrome is increasing rapidly and is estimated to affect 24% of the United States population (2). Thiazolidinediones, a class of antidiabetic compounds that activate the peroxisome proliferator-activated receptor-␥ (PPAR␥), 1 stimulate adipocyte differentiation, lower free fatty acids (FFAs), and enhance insulin sensitivity, thereby correcting several features of the metabolic syndrome (3). The insulin-sensitizing action of the thiazolidinediones suggests that PPAR␥ function may be central to the development and treatment of the metabolic syndrome.PPAR␥ is a member of the nuclear hormone receptor superfamily and plays a pivotal role in adipogenesis (4 -6). Although PPAR␥ is most highly expressed in adipose tissue, it is also present in colon, monocytes/macrophages, and at lower levels in many other tissues including skeletal muscle and liver (7,8). Homozygous PPAR␥ null mice die in utero at 10.5-11.5 days post-coitum, likely because of placental abnormalities (9). Heterozygous PPAR␥-deficient mice exhibit increased insulin sensitivity and are protected from high fat diet-induced obesity when compared with their wild-type littermates (10, 11). These observations may be partly explained by increased serum leptin, as heterozygous PPAR␥ knock-out mice have decreased food intake and increased energy expenditure (10). Cre-loxP strategies have been used to generate tissue-specific knockouts of PPAR␥ function (12-17). Muscle-specific PPAR␥ knockout mice show progressive insulin resistance combined with increased adipose tissue mass (14, 16). Fat-specific PPAR␥ knock-out mice have lipodystrophy (hypocellularity and hypertrophy), elevated plasma FFAs and triglycerides (TGs) and decreased plasma leptin and adiponectin. These mice have insulin resistance in fat and liver but not in muscle (13).Th...

Aims/hypothesis. PPARγ, a member of the nuclear hormone receptor family of transcription factors, plays a key role in adipocyte differentiation and insulin sensitivity. The aim of this study was to identify a potential dominant negative murine PPARγ mutant and to characterize the in vitro functional properties of this mutant. Methods. In vitro transient transfections and mammalian two-hybrid assays in TSA201 cells were used to characterize the transcriptional activity of the L466A mutant and to study the molecular interaction of transcriptional cofactors with the L466A mutant in an attempt to elucidate the mechanism of its dominant negative activity. Adenoviral constructs expressing PPARγ wild-type (AdWT) or the L466A mutant (AdL466A) were infected into the murine 3T3-L1 cell line to study the mutant's effect on adipogenesis. Results. The L466A mutant alone is transcriptionally defective. However, it retains DNA binding and inhibits the ligand-dependent and -independent activity of the wild-type receptor, consistent with dominant negative properties. In mammalian two-hybrid studies, the L466A mutant does not bind nuclear receptor coactivators. However, it more avidly recruits corepressors due to enhanced binding to the corepressor ID1 domain, leading to pronounced transcriptional repression. The AdL466A mutant inhibits adipogenesis induced by either a differentiation cocktail or by thiazolidinedione ligand. AdL466A infection also blocked the upregulation of the adipocyte marker genes aP2 and adipsin. Conclusion. We conclude that the L466A PPARγ mutant possesses potent dominant negative activity based on preferential corepressor recruitment and it inhibits adipogenesis and the expression of adipocyte-specific genes. [Diabetologia (2003) 46:365-377]