Timothy M. Willson

The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation

The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen. PPAR-γ has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) bind to PPAR-γ and stimulate transcription of target genes. Prostaglandin D2metabolites have not yet been identified in adipose tissue, butaremajor products of arachidonic-acid metabolism in macrophages, raising the possibility that they might serve as endogenous PPAR-γ ligands in this cell type. Here we show that PPAR-γ is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-γ ligands. PPAR-γ inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-κB. These observations suggest that PPAR-γ and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-γ ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.

A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation

Prostaglandins (PGs) of the J2 series form in vivo and exert effects on a variety of biological processes. While most of PGs mediate their effects through G protein-coupled receptors, the mechanism of action for the J2 series of PGs remains unclear. Here, we report the PGJ2 and its derivatives are efficacious activators of peroxisome proliferator-activated receptors alpha and gamma (PPAR alpha and PPAR gamma, respectively), orphan nuclear receptors implicated in lipid homeostasis and adipocyte differentiation. The PGJ2 metabolite 15-deoxy-delta 12,14-PGJ2 binds directly to PPAR gamma and promotes efficient differentiation of C3H10T1/2 fibroblasts to adipocytes. These data provide strong evidence that a fatty acid metabolite can function as an adipogenic agent through direct interactions with PPAR gamma and furthermore, suggest a novel mechanism of action for PGs of the J2 series.

Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma.

The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-γ ligand-binding domain (LBD), at 2.2 Å resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-γ. We also describe the ternary complex containing the PPAR-γ LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 Å resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a ‘charge clamp’ that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-γ homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.

The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions.

The cytochrome P-450 monooxygenase 3A4 (CYP3A4) is responsible for the oxidative metabolism of a wide variety of xenobiotics including an estimated 60% of all clinically used drugs. Although expression of the CYP3A4 gene is known to be induced in response to a variety of compounds, the mechanism underlying this induction, which represents a basis for drug interactions in patients, has remained unclear. We report the identification of a human (h) orphan nuclear receptor, termed the pregnane X receptor (PXR), that binds to a response element in the CYP3A4 promoter and is activated by a range of drugs known to induce CYP3A4 expression. Comparison of hPXR with the recently cloned mouse PXR reveals marked differences in their activation by certain drugs, which may account in part for the species-specific effects of compounds on CYP3A gene expression. These findings provide a molecular explanation for the ability of disparate chemicals to induce CYP3A4 levels and, furthermore, provide a basis for developing in vitro assays to aid in predicting whether drugs will interact in humans.

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.

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.

A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.

Bile acids repress the transcription of cytochrome P450 7A1 (CYP7A1), which catalyzes the rate-limiting step in bile acid biosynthesis. Although bile acids activate the farnesoid X receptor (FXR), the mechanism underlying bile acid-mediated repression of CYP7A1 remained unclear. We have used a potent, nonsteroidal FXR ligand to show that FXR induces expression of small heterodimer partner 1 (SHP-1), an atypical member of the nuclear receptor family that lacks a DNA-binding domain. SHP-1 represses expression of CYP7A1 by inhibiting the activity of liver receptor homolog 1 (LRH-1), an orphan nuclear receptor that is known to regulate CYP7A1 expression positively. This bile acid-activated regulatory cascade provides a molecular basis for the coordinate suppression of CYP7A1 and other genes involved in bile acid biosynthesis.

Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7α-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRα and LXRβ, 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 7α-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.

A selective peroxisome proliferator-activated receptor δ agonist promotes reverse cholesterol transport

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the antidiabetic effects of the glitazone drugs in humans are due to activation of the α (NR1C1) and γ (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the δ (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPARδ agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPARδ agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.

A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has essential roles in adipogenesis and glucose homeostasis, and is a molecular target of insulin-sensitizing drugs. Although the ability of PPAR-γ agonists to antagonize inflammatory responses by transrepression of nuclear factor kappa B (NF-κB) target genes is linked to antidiabetic and antiatherogenic actions, the mechanisms remain poorly understood. Here we report the identification of a molecular pathway by which PPAR-γ represses the transcriptional activation of inflammatory response genes in mouse macrophages. The initial step of this pathway involves ligand-dependent SUMOylation of the PPAR-γ ligand-binding domain, which targets PPAR-γ to nuclear receptor corepressor (NCoR)–histone deacetylase-3 (HDAC3) complexes on inflammatory gene promoters. This in turn prevents recruitment of the ubiquitylation/19S proteosome machinery that normally mediates the signal-dependent removal of corepressor complexes required for gene activation. As a result, NCoR complexes are not cleared from the promoter and target genes are maintained in a repressed state. This mechanism provides an explanation for how an agonist-bound nuclear receptor can be converted from an activator of transcription to a promoter-specific repressor of NF-κB target genes that regulate immunity and homeostasis.

Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.