Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR.

The orphan nuclear receptor SXR coordinately regulates drug clearance in response to a wide variety of xenobiotic compounds. This signaling system protects the body from exposure to toxic compounds; however, it can also pose a severe barrier to drug therapy. We now demonstrate that the human immunodeficiency virus (HIV) protease inhibitor ritonavir binds SXR and activates its target genes. This represents an example of a commonly used therapeutic agent that effectively activates SXR. We also show that other protease inhibitors are weaker (saquinavir) or unable to activate SXR (nelfinavir, indinavir) thus defining analogs that fail to induce SXR-regulated clearance pathways. Interestingly, HIV protease inhibitors are distinct from previously known SXR ligands in that they are peptide mimetic compounds. This expands the ligand specificity of SXR to include this unique chemical class whose pharmaceutical significance is expanding. Finally, we show that SXR ligands activate expression of multiple resistance protein 2, a critical regulator of bile flow and biliary drug excretion. These findings have important implications for the role of SXR in regulating drug clearance and hepatic disorders associated with impaired bile flow.

The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux.

Cytochrome P450 3A4 is an important mediator of drug catabolism that can be regulated by the steroid and xenobiotic receptor (SXR). We show here that SXR also regulates drug efflux by activating expression of the gene MDR1, which encodes the protein P-glycoprotein (ABCB1). Paclitaxel (Taxol), a commonly used chemotherapeutic agent, activated SXR and enhanced P-glycoprotein-mediated drug clearance. In contrast, docetaxel (Taxotere), a closely related antineoplastic agent, did not activate SXR and displayed superior pharmacokinetic properties. Docetaxel's silent properties reflect its inability to displace transcriptional corepressors from SXR. We also found that ET-743, a potent antineoplastic agent, suppressed MDR1 transcription by acting as an inhibitor of SXR. These findings demonstrate how the molecular activities of SXR can be manipulated to control drug clearance.

Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.

The major metabolic pathway for elimination of cholesterol is via conversion to bile acids. In addition to this metabolic function, bile acids also act as signaling molecules that negatively regulate their own biosynthesis. However, the precise nature of this signaling pathway has been elusive. We have isolated an endogenous biliary component (chenodeoxycholic acid) that selectively activates the orphan nuclear receptor, FXR. Structure-activity analysis defined a subset of related bile acid ligands that activate FXR and promote coactivator recruitment. Finally, we show that ligand-occupied FXR inhibits transactivation from the oxysterol receptor LXR alpha, a positive regulator of cholesterol degradation. We suggest that FXR (BAR) is the endogenous bile acid sensor and thus an important regulator of cholesterol homeostasis.

Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta.

The orphan receptor CAR-beta binds DNA as a heterodimer with the retinoid-X receptor and activates gene transcription in a constitutive manner. Here we show that, in contrast to the classical nuclear receptors, the constitutive activity of CAR-beta results from a ligand-independent recruitment of transcriptional co-activators. While searching for potential ligands of CAR-beta, we found that the steroids androstanol and androstenol inhibit the constitutive activity of CAR-beta. This effect is stereospecific: only 3alpha-hydroxy, 5alpha-reduced androstanes are active. These androstanes do not interfere with heterodimerization or DNA binding of CAR-beta; instead, they promote co-activator release from the ligand-binding domain. These androstane ligands are examples of naturally occurring inverse agonists that reverse transcriptional activation by nuclear receptors. CAR-beta (constitutive androstane receptor-beta), therefore, defines an unanticipated steroidal signalling pathway that functions in a manner opposite to that of the conventional nuclear receptor pathways.

The orphan nuclear receptor LXRalpha is positively and negatively regulated by distinct products of mevalonate metabolism.

LXRalpha is an orphan member of the nuclear hormone receptor superfamily that displays constitutive transcriptional activity. We reasoned that this activity may result from the production of an endogenous activator that is a component of intermediary metabolism. The use of metabolic inhibitors revealed that mevalonic acid biosynthesis is required for LXRalpha activity. Mevalonic acid is a common metabolite used by virtually all eukaryotic cells. It serves as a precursor to a large number of important molecules including farnesyl pyrophosphate, geranylgeranyl pyrophosphate, cholesterol, and oxysterols. Inhibition of LXRalpha could be reversed by addition of mevalonic acid and certain oxysterols but not by other products of mevalonic acid metabolism. Surprisingly, the constitutive activity of LXRalpha was inhibited by geranylgeraniol, a metabolite of mevalonic acid. These findings suggest that LXRalpha may represent a central component of a signaling pathway that is both positively and negatively regulated by multiple products of mevalonate metabolism.

Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta.

Fatty acids (FAs) and their derivatives are essential cellular metabolites whose concentrations must be closely regulated. This implies that regulatory circuits exist which can sense changes in FA levels. Indeed, the peroxisome proliferator-activated receptor alpha (PPARalpha) regulates lipid homeostasis and is transcriptionally activated by a variety of lipid-like compounds. It remains unclear as to how these structurally diverse compounds can activate a single receptor. We have developed a novel conformation-based assay that screens activators for their ability to bind to PPARalpha/delta and induce DNA binding. We show here that specific FAs, eicosanoids, and hypolipidemic drugs are ligands for PPARalpha or PPARdelta. Because altered FA levels are associated with obesity, atherosclerosis, hypertension, and diabetes, PPARs may serve as molecular sensors that are central to the development and treatment of these metabolic disorders.

Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor gamma and the retinoid X receptor.

Induction of terminal differentiation represents a promising therapeutic approach to certain human malignancies. The peroxisome proliferator-activated receptor gamma (PPAR gamma) and the retinoid X receptor alpha (RXR alpha) form a heterodimeric complex that functions as a central regulator of adipocyte differentiation. Natural and synthetic ligands for both receptors have been identified. We demonstrate here that PPAR gamma is expressed at high levels in each of the major histologic types of human liposarcoma. Moreover, primary human liposarcoma cells can be induced to undergo terminal differentiation by treatment with the PPAR gamma ligand pioglitazone, suggesting that the differentiation block in these cells can be overcome by maximal activation of the PPAR pathway. We further demonstrate that RXR-specific ligands are also potent adipogenic agents in cells expressing the PPAR gamma/RXR alpha heterodimer, and that simultaneous treatment of liposarcoma cells with both PPAR gamma- and RXR-specific ligands results in an additive stimulation of differentiation. Liposarcoma cell differentiation is characterized by accumulation of intracellular lipid, induction of adipocyte-specific genes, and withdrawal from the cell cycle. These results suggest that PPAR gamma ligands such as thiazolidinediones and RXR-specific retinoids may be useful therapeutic agents for the treatment of liposarcoma.

The peroxisome proliferator-activated receptors: ligands and activators.

PPAR alpha and PPAR gamma represent related but distinct members of the nuclear receptor superfamily. PPAR alpha signaling is modulated by long-chain fatty acids, whereas PPAR gamma ligands are potent antidiabetic agents.

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.

Two distinct dimerization interfaces differentially modulate target gene specificity of nuclear hormone receptors.

Several nuclear receptors including the all-trans retinoic acid receptor RAR, form heterodimers with the 9-cis retinoic acid receptor, RXR. RXR-RAR heterodimers show an impressive flexibility in DNA binding and can recognize palindromic, inverted palindromes and direct repeats of the core half-site sequence AGGTCA. Dimerization interfaces in the DNA-binding domains of RXR, RAR, and thyroid hormone receptor (TR) that promote selective binding to strictly spaced direct repeats have previously been identified. However, an additional dimerization domain is present within the ligand-binding domains (LBDs) of these receptors. Here we localize a transferable 40-amino acid region within the LBDs of RXR, RAR, TR, and chicken ovalbumin upstream promoter transcription factor that is critical for determining identity in the heterodimeric interaction and for high-affinity DNA binding. This region overlaps almost perfectly with a helical segment in the RXR LBD crystal structure that was recently demonstrated to be part of the dimer interface. Our data suggest a sequential pathway for nuclear receptor dimerization whereby the LBD dimerization interface initiates the formation of solution heterodimers that, in turn, acquire the capacity to bind to a number of differently organized repeats. Formation of a second dimer interface within the DNA-binding domain (DBD) restricts receptors to direct repeat targets. Accordingly, the combination of an obligatory (LBD) and an optional (DBD) dimerization domain imparts a dynamic DNA-binding potential to the heterodimerizing receptors that both increases the diversity of the hormonal response as well as providing a restricted set of target sequences in direct repeat elements that ensures physiological specificity.

Differential activation of adipogenesis by multiple PPAR isoforms.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear hormone receptor expressed predominantly in adipose tissue, where it plays a central role in the control of adipocyte gene expression and differentiation. Because there are two additional PPAR isoforms, PPARalpha and PPARdelta, and these are also expressed at some level in certain adipose depots, we have compared directly the adipogenic potential of all three receptors. Ectopically expressed PPARgamma powerfully induces adipogenesis at a morphological and molecular level in response to a number of PPARgamma activators. PPARalpha is less adipogenic but is able to induce significant differentiation in response to strong PPARalpha activators. Expression and activation of PPARdelta did not stimulate adipogenesis. Of the three PPARs, only PPARgamma can cooperate with C/EBPalpha in the promotion of adipogenesis. To begin to investigate the functional basis for the differential adipogenic activity of the PPAR isoforms, we have examined their ability to bind to several PPAR DNA response sequences. Compared with PPARalpha and PPARdelta, PPARgamma shows preferential binding to two well-characterized regulatory sequences derived from a fat-specific gene, ARE6 and ARE7. These data strongly suggest that PPARgamma is the predominant receptor regulating adipogenesis; however, they also suggest that PPARalpha may play a role in differentiation of certain adipose depots in response to a different set of physiologic activators or in certain disease states.

15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma.

Regulation of adipose cell mass is a critical homeostatic process in higher vertebrates. The conversion of fibroblasts into cells of the adipose lineage is induced by expression of the orphan nuclear receptor PPAR gamma. This suggests that an endogenous PPAR gamma ligand may be an important regulator of adipogenesis. By assaying arachidonate metabolites for their capacity to activate PPAR response elements, we have identified 15-deoxy-delta 12, 14-prostaglandin J2 as both a PPAR gamma ligand and an inducer of adipogenesis. Similarly, the thiazolidinedione class of antidiabetic drugs also bind to PPAR gamma and act as potent regulators of adipocyte development. Thus, adipogenic prostanoids and antidiabetic thiazolidinediones initiate key transcriptional events through a common nuclear receptor signaling pathway. These findings suggest a pivotal role for PPAR gamma and its endogenous ligand in adipocyte development and glucose homeostasis and as a target for intervention in metabolic disorders.

Identification of a nuclear receptor that is activated by farnesol metabolites.

Nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that mediate the transcriptional activities of steroids, retinoids, and thyroid hormones. A growing number of related proteins have been identified that possess the structural features of hormone receptors, but that lack known ligands. Known as orphan receptors, these proteins represent targets for novel signaling molecules. We have isolated a mammalian orphan receptor that forms a heterodimeric complex with the retinoid X receptor. A screen of candidate ligands identified farnesol and related metabolites as effective activators of this complex. Farnesol metabolites are generated intracellularly and are required for the synthesis of cholesterol, bile acids, steroids, retinoids, and farnesylated proteins. Intermediary metabolites have been recognized as transcriptional regulators in bacteria and yeast. Our results now suggest that metabolite-controlled intracellular signaling systems are utilized by higher organisms.

Nuclear hormone receptors activate direct, inverted, and everted repeats.

Nuclear hormone receptors comprise a family of ligand-modulated transcription factors that link cellular responses to extracellular and intracellular signals. Receptors for retinoids, thyroid hormone, vitamin D3 and fatty acids/peroxisome proliferators bind their response elements as heterodimers with the retinoid X receptor. Naturally occurring response elements are composed of core-motifs that are organized as direct, inverted, and/or everted repeats. The structural mechanisms that facilitate binding of a single receptor heterodimer to such diverse binding sites remain unknown.

Unique response pathways are established by allosteric interactions among nuclear hormone receptors.

Heterodimerization is a common paradigm among eukaryotic transcription factors. The 9-cis retinoic acid receptor (RXR) serves as a common heterodimerization partner for several nuclear receptors, including the thyroid hormone receptor (T3R) and retinoic acid receptor (RAR). This raises the question as to whether these complexes possess dual hormonal responsiveness. We devised a strategy to examine the transcriptional properties of each receptor individually or when tethered to a heterodimeric partner. We find that the intrinsic binding properties of RXR are masked in T3R-RXR and RAR-RXR heterodimers. In contrast, RXR is active as a non-DNA-binding cofactor with the NGFI-B/Nurr1 orphan receptors. Heterodimerization of RXR with constitutively active NGFI-B/Nurr1 creates a novel hormone-dependent complex. These findings suggest that allosteric interactions among heterodimers create complexes with unique properties. We suggest that allostery is a critical feature underlying the generation of diversity in hormone response networks.

Cross-talk among ROR alpha 1 and the Rev-erb family of orphan nuclear receptors.

We have cloned Rev-erb beta, a novel isoform of the Rev-erb alpha orphan nuclear receptor. The DNA binding domains of Rev-erb alpha and beta are highly related to each other and to the retinoic acid related orphan receptor (ROR)/RZR subfamily of nuclear receptors. Indeed, we find that all three receptors bind as monomers to the sequence AATGT-AGGTCA. Whereas ROR alpha 1 constitutively activates transcription through this sequence, both isoforms of Rev-erb are inactive. When coexpressed, both Rev-erb isoforms suppress the transcriptional activity of ROR alpha 1. Our data define Rev-erb and ROR/RZR as a family of related receptors with opposing activities on overlapping regulatory networks.

Differential expression and activation of a family of murine peroxisome proliferator-activated receptors.

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in mammals, we have cloned and characterized two PPAR alpha-related cDNAs (designated PPAR gamma and -delta, respectively) from mouse. The three PPAR isoforms display widely divergent patterns of expression during embryogenesis and in the adult. Surprisingly, PPAR gamma and -delta are not activated by pirinixic acid (Wy 14,643), a potent peroxisome proliferator and activator of PPAR alpha. However, PPAR gamma and -delta are activated by the structurally distinct peroxisome proliferator LY-171883 and linoleic acid, respectively, indicating that each of the isoforms can act as a regulated activator of transcription. These data suggest that tissue-specific responsiveness to peroxisome proliferators, including certain fatty acids, is in part a consequence of differential expression of multiple, pharmacologically distinct PPAR isoforms.