Knut R. Steffensen

Activated Liver X Receptors Stimulate Adipocyte Differentiation through Induction of Peroxisome Proliferator-Activated Receptor γ Expression

ABSTRACT Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARγ (peroxisome proliferator-activated receptor γ) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARγ gene is a novel target gene of LXR, since the PPARγ promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRα. Moreover, activated LXRα exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARγ, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRα by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.

Liver X receptor biology and pharmacology: new pathways, challenges and opportunities

Nuclear receptors (NRs) are master regulators of transcriptional programs that integrate the homeostatic control of almost all biological processes. Their direct mode of ligand regulation and genome interaction is at the core of modern pharmacology. The two liver X receptors LXRα and LXRβ are among the emerging newer drug targets within the NR family. LXRs are best known as nuclear oxysterol receptors and physiological regulators of lipid and cholesterol metabolism that also act in an anti-inflammatory way. Because LXRs control diverse pathways in development, reproduction, metabolism, immunity and inflammation, they have potential as therapeutic targets for diseases as diverse as lipid disorders, atherosclerosis, chronic inflammation, autoimmunity, cancer and neurodegenerative diseases. Recent insights into LXR signaling suggest future targeting strategies aiming at increasing LXR subtype and pathway selectivity. This review discusses the current status of our understanding of LXR biology and pharmacology, with an emphasis on the molecular aspects of LXR signaling that constitute the potential of LXRs as drug targets.

Accumulation of Foam Cells in Liver X Receptor-Deficient Mice

Background—The nature of some of the target genes for liver X receptors (LXRs)-&agr; and -&bgr;, such as sterol regulatory element binding protein-1 and ATP-binding cassette transporter proteins, suggests a pivotal role of these nuclear receptors in the regulation of fatty acid and cholesterol homeostasis. The present study aimed to elucidate the physiological relevance of both LXRs with regard to lipid metabolism and macrophage cholesterol efflux. Methods and Results—Mice depleted for LXR&agr;, LXR&bgr;, or both were fed low-fat rodent chow for 18 months before investigations. The combined deficiency of LXR&agr; and LXR&bgr; was linked to impaired triglyceride metabolism, increased LDL and reduced HDL cholesterol levels, and cholesterol accumulation in macrophages (foam cells) of the spleen, lung, and arterial wall. Conclusions—Our data demonstrate the physiological importance of both LXRs in lipid metabolism and strongly indicate that both LXRs have a protective role against the development of atherosclerosis.

Tumor-mediated liver X receptor-[alpha] activation inhibits CC chemokine receptor-7 expression on dendritic cells and dampens antitumor responses

Sterol metabolism has recently been linked to innate and adaptive immune responses through liver X receptor (LXR) signaling. Whether products of sterol metabolism interfere with antitumor responses is currently unknown. Dendritic cells (DCs) initiate immune responses, including antitumor activity after their CC chemokine receptor-7 (CCR7)-dependent migration to lymphoid organs. Here we report that human and mouse tumors produce LXR ligands that inhibit CCR7 expression on maturing DCs and, therefore, their migration to lymphoid organs. In agreement with this observation, we detected CD83+CCR7− DCs within human tumors. Mice injected with tumors expressing the LXR ligand–inactivating enzyme sulfotransferase 2B1b (SULT2B1b) successfully controlled tumor growth by regaining DC migration to tumor-draining lymph nodes and by developing overt inflammation within tumors. The control of tumor growth was also observed in chimeric mice transplanted with bone marrow from mice lacking the gene encoding LXR-α (Nr1h3−/− mice) Thus, we show a new mechanism of tumor immunoescape involving products of cholesterol metabolism. The manipulation of this pathway could restore antitumor immunity in individuals with cancer.

Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

ABSTRACT The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRβ in the hepatic acute phase response

The orphan receptor LRH-1 and the oxysterol receptors LXRalpha and LXRbeta are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRbeta subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor-corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

Glucocorticoid Signaling Is Perturbed by the Atypical Orphan Receptor and Corepressor SHP

SHP (NROB2) is an atypical orphan nuclear receptor that lacks a DNA-binding domain but contains a putative ligand-binding domain. Previous studies have revealed that SHP interacts with a variety of nuclear receptors and inhibits their transcriptional activity, thereby acting as a corepressor. In this report we identify the glucocorticoid receptor (GR) as a novel downstream target receptor for SHP inhibition. SHP potently inhibits dexamethasone-induced transcriptional GR activity in mammalian cells, and the inhibition involves a functional second NR-box within SHP. Interestingly, this motif shows a high homology with the NR-box in the glucocorticoid and cAMP-inducible GR coactivator PGC-1, indicating similar binding specificity and shared target receptors. We show that SHP antagonizes PGC-1 coactivation and, in addition, we identify the PGC- 1-regulated phospho(enol)pyruvate carboxykinase (PEPCK) promoter as a novel target promoter for SHP inhibition. This implies a physiologically relevant role for SHP in modulating hepatic glucocorticoid action. Furthermore, when coexpressing green fluorescent protein-tagged GR together with SHP, an intranuclear redistribution of GR was observed. As inhibition-deficient SHP mutants were unable to induce this redistribution, intranuclear tethering of target receptors may represent yet another, previously uncovered, aspect of SHP inhibition.

The oxysterol receptor LXR inhibits proliferation of human breast cancer cells

The oxysterol receptors [liver X receptors (LXRalpha and LXRbeta)] regulate cholesterol and lipid biosynthesis and several studies link dysregulation of these metabolic pathways to aberrant cell growth. Here, we show that activation of LXR significantly reduced proliferation in several human breast cancer cells lines. LXR suppressed messenger RNA and/or protein expression of Skp2, cyclin A2, cyclin D1 and estrogen receptor (ER) alpha, whereas it increased the expression of p53 at the protein level and maintained the retinoblastoma protein in a hypophosphorylated active form. These changes may constitute part of the molecular mechanisms behind the antiproliferative effect of LXR. Furthermore, activation of LXR induced expression of key lipogenic genes including sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase and stearoyl-coenzyme A desaturase 1, leading to increased triglyceride production in MCF7 cells. Small interfering RNA knockdown of SREBP1c, a master regulator of the lipid biosynthesis, did not abolish the antiproliferative effect of LXR in these cells. Combined these studies identify LXRs as both antiproliferative and lipogenic factors in breast cancer cells and indicate that the antiproliferative effect of LXRs is independent of lipid biosynthesis.

Liver X receptors contribute to the protective immune response against Mycobacterium tuberculosis in mice

Liver X receptors (LXRs) are key regulators of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation. However, exactly how LXRs modulate inflammation during infection remains unknown. To explore this, we used a mouse model of Mycobacterium tuberculosis infection. Upon intratracheal infection with M. tuberculosis, LXRs and LXR target genes were induced in CD11c+ lung and alveolar cells. Furthermore, mice deficient in both LXR isoforms, LXRalpha and LXRbeta (Lxra-/-Lxrb-/- mice), were more susceptible to infection, developing higher bacterial burdens and an increase in the size and number of granulomatous lesions. Interestingly, mice solely deficient in LXRalpha, but not those lacking only LXRbeta, mirrored the susceptibility of the Lxra-/-Lxrb-/- animals. Lxra-/-Lxrb-/- mice failed to mount an effective early neutrophilic airway response to infection and showed dysregulation of both pro- and antiinflammatory factors in CD11c+ lung cells. T cell responses were strongly affected in Lxra-/-Lxrb-/- mice, showing near-complete abrogation of the infection-induced Th1 function - and even more so Th17 function - in the lungs. Treatment of WT mice with the LXR agonists TO901317 and GW3965 resulted in a 10-fold decrease of the pulmonary bacterial burden and a comparable increase of Th1/Th17 function in the lungs. The dependence of LXR signaling on the neutrophil IL-17 axis represents what we believe to be a novel function for these nuclear receptors in resistance to M. tuberculosis infection and may provide a new target for therapeutics.

Liver X Receptors and Oxysterols Promote Ventral Midbrain Neurogenesis In Vivo and in Human Embryonic Stem Cells

Control over progenitor proliferation and neurogenesis remains a key challenge for stem cell neurobiology and a prerequisite for successful stem cell replacement therapies for neurodegenerative diseases like Parkinsons disease (PD). Here, we examined the function of two nuclear receptors, liver X receptors (Lxralpha and beta) and their ligands, oxysterols, as regulators of cell division, ventral midbrain (VM) neurogenesis, and dopaminergic (DA) neuron development. Deletion of Lxrs reduced cell cycle progression and VM neurogenesis, resulting in decreased DA neurons at birth. Activation of Lxrs with oxysterol ligands increased the number of DA neurons in mouse embryonic stem cells (ESCs) and in wild-type but not Lxralphabeta(-/-) VM progenitor cultures. Likewise, oxysterol treatment of human ESCs (hESCs) during DA differentiation increased neurogenesis and the number of mature DA neurons, while reducing proliferating progenitors. Thus, Lxr ligands may improve current hESC replacement strategies for PD by selectively augmenting the generation of DA neurons.