Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated by steroids. Notably, PXR.1 is efficaciously activated by pregnenolone 16alpha-carbonitrile, a glucocorticoid receptor antagonist that induces the expression of the CYP3A family of steroid hydroxylases and modulates sterol and bile acid biosynthesis in vivo. Our results provide evidence for the existence of a novel steroid hormone signaling pathway with potential implications in the regulation of steroid hormone and sterol homeostasis.

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7␣-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXR␣ and LXR␤, two orphan members of the nuclear receptor superfamily, are activated by 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7␣-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis.Cholesterol (CH) 1 is a major structural constituent of cellular membranes and serves as the biosynthetic precursor for bile acids and steroid hormones. Animal cells can obtain CH endogenously through de novo synthesis from acetyl-CoA or exogenously through receptor-mediated endocytosis of low density lipoproteins. Cells must balance the internal and external sources of CH so as to maintain mevalonate biosynthesis while at the same time avoiding the accumulation of excess CH, which can result in diseases such as atherosclerosis, gallstones, and several lipid storage disorders (1).CH homeostasis is maintained in part through feedback regulation of the low density lipoprotein receptor gene and at least two genes encoding enzymes in the CH biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase (1). Although increases in dietary CH lead to the inhibition of expression of these genes in vivo, it remains unclear whether CH or CH metabolites are responsible for this inhibition (2). Experiments performed in vitro using several different cell lines have indicated that derivatives of CH that are oxygenated on the CH side chain are significantly more potent in the suppression of sterol biosynthesis than CH (3). These oxysterols are produced through the actions of P450 enzymes in various metabolic pathways including bile acid synthesis in the liver and sex hormone synthesis in the adrenal glands. The in vitro activities of oxysterols together with their presence in vivo suggests that oxysterols may serve in metabolic feedback loops to regulate CH homeostasis.Although CH and its oxysterol metabolites can repress gene transcription, in at least one instance dietary CH has been shown to stimulate gene expression. Expression of the cholesterol 7␣-hydroxylase (CYP7A) gene, which encodes the enzyme responsible for the initial and rate-limiting step in the conversion of CH to bile acids (4), is up-regulated in rats fed a CH-rich diet (5-7). This stimulatory effect provides a regulatory mechanism whereby excess dietary CH can be converted to more polar bile acids for subsequent removal from the body. Although the molecular mechanism is unknown, induction of CYP7A expression in the presence of CH occurs at the level...

Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) and cyclooxygenase inhibitor that is frequently used as a research tool to study the process of adipocyte differentiation. Treatment of various preadipocyte cell lines with micromolar concentrations of indomethacin in the presence of insulin promotes their terminal differentiation. However, the molecular basis for the adipogenic actions of indomethacin had remained unclear. In this report, we show that indomethacin binds and activates peroxisome proliferatoractivated receptor ␥ (PPAR␥), a ligand-activated transcription factor known to play a pivotal role in adipogenesis. The concentration of indomethacin required to activate PPAR␥ is in good agreement with that required to induce the differentiation of C3H10T1/2 cells to adipocytes. We demonstrate that several other NSAIDs, including fenoprofen, ibuprofen, and flufenamic acid, are also PPAR␥ ligands and induce adipocyte differentiation of C3H10T1/2 cells. Finally, we show that the same NSAIDs that activate PPAR␥ are also efficacious activators of PPAR␣, a liver-enriched PPAR subtype that plays a key role in peroxisome proliferation. Interestingly, several NSAIDs have been reported to induce peroxisomal activity in hepatocytes both in vitro and in vivo. Our findings define a novel group of PPAR␥ ligands and provide a molecular basis for the biological effects of these drugs on adipogenesis and peroxisome activity.Indomethacin and other NSAIDs 1 are used clinically for their anti-inflammatory, anti-pyretic, and analgesic properties (1). The molecular basis for the therapeutic actions of NSAIDs is believed to be their ability to inhibit cyclooxygenase (COX)

Brown adipose tissue (BAT) functions in non-shivering and diet-induced thermogenesis via its capacity for uncoupled mitochondrial respiration. BAT dysfunction in rodents is associated with severe defects in energy homeostasis, resulting in obesity and hyperglycemia. Here, we report that the nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥), a prostaglandin-activated transcription factor recently implicated as a central regulator of white adipose tissue differentiation, also regulates brown adipocyte function. PPAR␥ is abundantly expressed in both embryonic and adult BAT. Treatment of CD-1 rats with the PPAR␥-selective ligand BRL49653, an anti-diabetic drug of the thiazolidinedione class, results in marked increases in the mass of interscapular BAT. In vitro, BRL49653 induces the terminal differentiation of the brown preadipocyte cell line HIB-1B as judged by both changes in cell morphology and expression of uncoupling protein and other adipocyte-specific mRNAs. These data demonstrate that PPAR␥ is a key regulatory factor in brown adipocytes and suggest that PPAR␥ functions not only in the storage of excess energy in white adipose tissue but also in its dissipation in BAT.Two types of adipose tissue have been described. White adipose tissue (WAT) 1 is specialized to store triglycerides and to release free fatty acids in response to changing energy requirements. A second type of adipose tissue, termed brown adipose tissue (BAT), is involved in the dissipation of energy via the generation of heat (see below). This unique thermogenic activity of BAT is tightly regulated and can be induced in response to either cold exposure or hyperphagia (1, 2). In rodents, several lines of evidence suggest that BAT plays a central role in maintaining energy balance. First, by increasing energy expenditure in response to increased food intake, BAT thermogenesis acts to prevent (or deter) the development of obesity (3). Second, transgenic ablation of BAT in mice is sufficient to induce obesity as well as insulin resistance and other metabolic disorders that, as a whole, closely resemble human non-insulindependent diabetes mellitus (5-7). Third, defects in BAT function are thought to play a significant role in the development of obesity and diabetes in several animal models (8,9). These data suggest a tight link between BAT function and the regulation of glucose and lipid metabolism.The unique thermogenic activity of BAT results from the action of a BAT-specific protein termed uncoupling protein (UCP). UCP is a mitochondrial proton translocator that uncouples fatty acid oxidation from ATP synthesis, releasing the energy as heat (10, 11). Analysis of the UCP gene has resulted in the identification of a 220-bp enhancer located from Ϫ2.5 to Ϫ2.3 kilobase pairs upstream of the UCP gene that is responsible for brown adipocyte-specific gene expression in cell culture models (12,13). This enhancer region contains a cAMP response element as well as thyroid hormone receptor and retinoid receptor response elements (12-15)...

The female sex hormone estradiol (1) has a variety of beneficial and detrimental effects in women.' The triphenylethylene class of non-steroidal estrogens (e.g., tamoxifen, 2) shows tissue-dependent expression of estrogen agonist and antagonist activity and may represent a significant advance over conventional hormone replacement therapy with 1 for prevention of osteoporosis and cardiovascular disease in postmenopausal women2 ( Figure 1). The estrogen receptor is a ligand-activated transcription factor that belongs to the steroidbetinoid family of DNA-binding intracellular receptors (ICR). Studies with deletion and point-mutated receptors have revealed two independent transcription activation domains (AF-1 and AF-2, Figure 2) within the receptor that allow the expression of cell-and promoter-specific agonist activity in transient cotransfection experiments in vitr0.3 The translation of these observations to the design of ligands for ICRs that show tissue-specific expression of functional activity is at the forefront of modern endo~rinology.~ For this purpose, we formulated the hypothesis that the tissueselective profile of 2 was due to induction of a unique receptor conformation5 in which the antagonist activity in some tissues was due to disruption of AF-2, mediated by a H-bond interaction6 with the receptor protein in the region of the putative AF-2 a -h e l i~,~~~~ and the agonist activity in other tissues was a result of a functional AF-1 domain.3bld Combining this hypothesis with analysis of the in vitro and in vivo pharmacology of non-steroidal estrogens: it was proposed that the stilbene portion of 2 was required for AF-1 activity leading to agonist activity in bone, and the ethanolamine side chain was responsible for blocking AF-2 activity leading to antagonism in the uterus. We report here on the use of this hypothesis to identify triphenylethylene estrogens that show full agonist activity in bone through inhibition of bone loss in ovariectomized rats but which are antagonists in the rat uterus with minimal residual agonist activity.We elected to synthesize analogs of the triphenylethylene 2 in which the ethanolamine side chain was replaced by alternate H-bond acceptor groups and the degree of conformational freedom was reduced. Following the general synthetic strategy of Millerg for synthesis of (2)-tamoxifen, bromide 3 was coupled with arylboronic acid