Enrique Saez

ACTIVATORS OF THE NUCLEAR RECEPTOR PPARGAMMA ENHANCE COLON POLYP FORMATION

A high-fat diet increases the risk of colon, breast and prostate cancer. The molecular mechanism by which dietary lipids promote tumorigenesis is unknown. Their effects may be mediated at least in part by the peroxisome proliferator-activated receptors (PPARs). These ligand-activated nuclear receptors modulate gene expression in response to fatty acids, lipid-derived metabolites and antidiabetic drugs. To explore the role of the PPARs in diet-induced carcinogenesis, we treated mice predisposed to intestinal neoplasia with a synthetic PPARγ ligand. Reflecting the pattern of expression of PPARγ in the gastrointestinal tract, treated mice developed a considerably greater number of polyps in the colon but not in the small intestine, indicating that PPARγ activation may provide a molecular link between a high-fat diet and increased risk of colorectal cancer.

Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue

The control of lipid and glucose metabolism is closely linked. The nuclear receptors liver X receptor (LXR)α and LXRβ have been implicated in gene expression linked to lipid homeostasis; however, their role in glucose metabolism is not clear. We demonstrate here that the synthetic LXR agonist GW3965 improves glucose tolerance in a murine model of diet-induced obesity and insulin resistance. Analysis of gene expression in LXR agonist-treated mice reveals coordinate regulation of genes involved in glucose metabolism in liver and adipose tissue. In the liver, activation of LXR led to the suppression of the gluconeogenic program including down-regulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase expression. Inhibition of gluconeogenic genes was accompanied by an induction in expression of glucokinase, which promotes hepatic glucose utilization. In adipose tissue, activation of LXR led to the transcriptional induction of the insulin-sensitive glucose transporter, GLUT4. We show that the GLUT4 promoter is a direct transcriptional target for the LXR/retinoid X receptor heterodimer and that the ability of LXR ligands to induce GLUT4 expression is abolished in LXR null cells and animals. Consistent with their effects on GLUT4 expression, LXR agonists promote glucose uptake in 3T3-L1 adipocytes in vitro. Thus, activation of LXR alters the expression of genes in liver and adipose tissue that collectively would be expected to limit hepatic glucose output and improve peripheral glucose uptake. These results outline a role for LXRs in the coordination of lipid and glucose metabolism.

The nuclear receptor LXR is a glucose sensor

The liver has a central role in glucose homeostasis, as it has the distinctive ability to produce and consume glucose. On feeding, glucose influx triggers gene expression changes in hepatocytes to suppress endogenous glucose production and convert excess glucose into glycogen or fatty acids to be stored in adipose tissue. This process is controlled by insulin, although debate exists as to whether insulin acts directly or indirectly on the liver. In addition to stimulating pancreatic insulin release, glucose also regulates the activity of ChREBP, a transcription factor that modulates lipogenesis. Here we describe another mechanism whereby glucose determines its own fate: we show that glucose binds and stimulates the transcriptional activity of the liver X receptor (LXR), a nuclear receptor that coordinates hepatic lipid metabolism. d-Glucose and d-glucose-6-phosphate are direct agonists of both LXR-α and LXR-β. Glucose activates LXR at physiological concentrations expected in the liver and induces expression of LXR target genes with efficacy similar to that of oxysterols, the known LXR ligands. Cholesterol homeostasis genes that require LXR for expression are upregulated in liver and intestine of fasted mice re-fed with a glucose diet, indicating that glucose is an endogenous LXR ligand. Our results identify LXR as a transcriptional switch that integrates hepatic glucose metabolism and fatty acid synthesis.

LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

c-fos is required for malignant progression of skin tumors

The proto-oncogene c-fos is a major nuclear target for signal transduction pathways involved in the regulation of cell growth, differentiation, and transformation. Using the multistep skin carcinogenesis model, we have directly tested the ability of c-fos-deficient mice to develop cancer. Upon treatment with a tumor promoter, c-fos knockout mice carrying a v-H-ras transgene were able to develop benign tumors with similar kinetics and relative incidence as wild-type animals. However, c-fos-deficient papillomas quickly became very dry and hyperkeratinized, taking on an elongated, horny appearance. While wild-type papillomas eventually progressed into malignant tumors, c-fos-deficient tumors failed to undergo malignant conversion. Experiments in which v-H-ras-expressing keratinocytes were grafted onto nude mice suggest that c-fos-deficient cells have an intrinsic defect that hinders tumorigenesis. These results demonstrate that a member of the AP-1 family of transcription factors is required for the development of a malignant tumor.

T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR.

The liver X receptors (LXRα and β) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Insight into the physiologic roles of the LXRs has been greatly facilitated by the discovery of potent synthetic agonists. Here we show that one of these compounds, T0901317, is also a high‐affinity ligand for the xenobiotic receptor pregnane X receptor (PXR). T0901317 binds and activates PXR with the same nanomolar potency with which it stimulates LXR activity. T0901317 induces expression not only of LXR target genes, but also of PXR target genes in cells and animals, including the scavenger receptor CD36, a property not shared by more specific LXR ligands, such as GW3965. Activation of PXR targets may explain why T0901317 induces dramatic liver steatosis, while GW3965 has a milder effect. These results suggest that many of the biological activities heretofore associated with LXR activation may be mediated by PXR, not LXR. Since T0901317 has been widely used in animals to study LXR function, the in vivo effects of this compound ascribed to LXR activation should be re‐examined.

Inhibition of Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances Oxidative Metabolism in Skeletal Muscle and Adipose Tissue

Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I–specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I– but not a class II–selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I–selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.

“Don't Know Much Bile-ology”

The work discussed above and an additional paper strengthen the notion that nuclear receptors are key regulators of cholesterol catabolism. The paper by Repa et al. links RXRs and LXRs to pathways of cholesterol absorption and efflux (Repa et al. 2000xRepa, J.J, Turley, S.D, Lobaccaro, J.A, Medina, J, Li, L, Lustig, K, Shan, B, Heyman, R.A, Dietschy, J.M, and Mangelsdorf, D.J. Science. 2000; 289: 1524–1529Crossref | PubMed | Scopus (953)See all ReferencesRepa et al. 2000). These authors found that treatment of mice with the RXR ligand, LG268, results in potent inhibition of cholesterol absorption. A decrease in bile acid pool size and an induction of ABCA1, an ABC family transporter that mediates cholesterol efflux from cells, are each partly responsible for decreased cholesterol absorption. Furthermore, the LXR/RXR heterodimer specifically regulates the expression of ABCA1, since neither LG268 nor T0901317 (an LXR-specific agonist) can induce ABCA1 in LXRα/β double knockout mice. This body of work implicates nuclear receptors as the key regulators for cholesterol catabolism and efflux, and suggests that the cross talk between nuclear receptors may serve to regulate pathways of cholesterol and lipid homeostasis (Figure 1Figure 1).In the past few years, the perception of nuclear receptors as ligand-modulated transcription factors whose primary role is to mediate the effects of endocrine signals has been altered by the discovery of a multitude of orphan receptors that can respond to dietary components, products of intermediary metabolism, and xenobiotic chemicals (Blumberg and Evans 1998xBlumberg, B and Evans, R.M. Genes Dev. 1998; 12: 3149–3155Crossref | PubMedSee all ReferencesBlumberg and Evans 1998). The present work delineates the genetic hierarchy that regulates cholesterol and bile acid homeostasis: LXR α/β govern the uptake of dietary cholesterol by controlling its intestinal transporter, and the interactions among FXR, SHP, and LRH-1 form the basis for the coordinated regulation of cholesterol degradation and bile acid secretion. In macrophages and perhaps other tissues, expression of PPARγ, a target of known prescription drugs, establishes an additional layer of regulation of cholesterol efflux and absorption while RXR participates at all sites as a partner of PPARγ, LXR α/β, and FXR.As a complement to traditional cholesterol-lowering drugs that focus on inhibition of endogenous synthesis, the identification of this collection of nuclear receptors that mediates cholesterol absorption, reverse transport, and degradation provides an unprecedented opportunity to pharmacologically manipulate these natural cholesterol-controlling pathways. Each is a potentially interesting drug development target. RXR compounds have been shown to be effective insulin sensitizers as well as potent inhibitors of cholesterol absorption and bile acid synthesis in animals (Mukherjee et al. 1997xMukherjee, R, Davies, P.J, Crombie, D.L, Bischoff, E.D, Cesario, R.M, Jow, L, Hamann, L.G, Boehm, M.F, Mondon, C.E, Nadzan, A.M et al. Nature. 1997; 386: 407–410Crossref | PubMed | Scopus (495)See all ReferencesMukherjee et al. 1997). PPARγ ligands are already widely used in treatment of type II diabetes and more recently show promise in animals as inhibitors of atherosclerosis (Willson et al. 2000xWillson, T.M, Brown, P.J, Sternbach, D.D, and Henke, B.R. J. Med. Chem. 2000; 43: 527–550Crossref | PubMed | Scopus (1433)See all ReferencesWillson et al. 2000). Synthetic LXR agonists may lower serum cholesterol levels by decreasing intestinal absorption while preventing hepatic accumulation. FXR activators may be useful to treat disorders of bile acid metabolism. Although not yet identified, small molecules that regulate SHP and LRH-1 could be useful in cases of either hypercholesterolemia or cholestasis, though certainly much less is understood regarding the extent and specificity of their regulation. In spite of the tinkering that may be necessary to develop clinical weapons based on our new knowledge, the fact that at least six nuclear receptors regulate distinct aspects of cholesterol homeostasis means that new drugs controlling their activity will be discovered.*To whom correspondence should be addressed (e-mail: evans@salk.edu).

Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression

We describe a genome-wide gain-of-function screen for regulators of NF-κB, and identify Rap1 (Trf2IP), as an essential modulator of NF-κB-mediated pathways. NF-κB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IκB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-κB to make it transcriptionally competent. Rap1-mutant mice display defective NF-κB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-κB, and human breast cancers with NF-κB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-κB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.

Gene Set Enrichment in eQTL Data Identifies Novel Annotations and Pathway Regulators

Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on “trans-eQTL bands”, defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.