Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells.

Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver availability can limit even this therapy. Identification of novel therapeutics for genetic-based liver diseases requires models that provide measurable hepatic functions and phenotypes. Advances in stem cell and genome editing technologies could provide models for the investigation of cell-based genetic diseases, as well as the platforms for drug discovery. This report demonstrates a practical, and widely applicable, approach that includes the successful reprogramming of somatic cells from a patient with a urea cycle defect, their genetic correction and differentiation into hepatic organoids, and the subsequent demonstration of genetic and phenotypic change in the edited cells consistent with the correction of the defect. While individually rare, there is a large number of other genetic-based liver diseases. The approach described here could be applied to a broad range and a large number of patients with these hepatic diseases where it could serve as an in vitro model, as well as identify successful strategies for corrective cell-based therapy.

The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages.

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases.

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Generation of new hepatocyte-like in vitro models better resembling human lipid metabolism.

In contrast to human hepatocytes in vivo, which solely express acyl-coenzyme A:cholesterol acyltransferase (ACAT) 2, both ACAT1 and ACAT2 (encoded by SOAT1 and SOAT2) are expressed in primary human hepatocytes and in human hepatoma cell lines. Here, we aimed to create hepatocyte-like cells expressing the ACAT2, but not the ACAT1, protein to generate a model that - at least in this regard - resembles the human condition in vivo and to assess the effects on lipid metabolism. Using the Clustered Regularly Interspaced Short Palindromic Repeats technology, we knocked out SOAT1 in HepG2 and Huh7.5 cells. The wild type and SOAT2-only-cells were cultured with fetal bovine or human serum and the effects on lipoprotein and lipid metabolism were studied. In SOAT2-only-HepG2 cells, increased levels of cholesterol, triglycerides, apolipoprotein B and lipoprotein(a) in the cell media were detected; this was likely dependent of the increased expression of key genes involved in lipid metabolism (e.g. MTP, APOB, HMGCR, LDLR, ACACA, and DGAT2). Opposite effects were observed in SOAT2-only-Huh7.5 cells. Our study shows that the expression of SOAT1 in hepatocyte-like cells contributes to the distorted phenotype observed in HepG2 and Huh7.5 cells. As not only parameters of lipoprotein and lipid metabolism but also some markers of differentiation/maturation increase in the SOAT2-only-HepG2 cells cultured with HS, this cellular model represent an improved model for studies of lipid metabolism.

Lipidomic analysis of human primary hepatocytes following LXR activation with GW3965 identifies AGXT2L1 as a main target associated to changes in phosphatidylethanolamine.

Liver X receptor (LXR) agonists have the potential to alleviate obesity related diseases, particularly atherosclerosis. However, LXRs are transcriptional regulators that induce de novo lipogenesis and lipid accumulation in hepatocytes which represents a serious adverse effect. In this work, we sought to characterize the LXR agonist GW3965 effects on fatty acid (FA) and phospholipid (PL) remodelling and the correlation with gene expression in order to better understand the underlying effects leading to hepatic pathology upon LXR activation. Human primary hepatocytes treated for 48 h with GW3965 were analysed for changes in lipid metabolism gene expression by qPCR, variations in the FA profile was evaluated by GC-FID and in PL profiles using thin layer chromatography, ESI-MS and MS/MS analysis. Changes in cell membrane biochemical properties were studied using bilayer models generated with CHARMM-GUI. ELOLV6 and SCD1 mRNA increase was consistent with higher C16:1 and C18:1n9 at the expense of C16:0 and C18:0. The reduction of C18:2n6 and increase in C20:2n6 was in agreement with ELOVL5 upregulation. Phosphatydilethanolamine (PE) levels tended to decrease and phosphatidylinositol to increase; although differences did not reach significance, they correlated with changes in AGXT2L1, CDS1 and LPIN1 mRNA levels that were increased. The overall effect of GW3965 on PEs molecular profiles was an increase of long-chain polyunsaturated FA chains and a decrease of C16/C18 saturated and monounsaturated FAs chains. Additionally, PC (32:1) and PC (34:2) were decreased, and PC (36:1) and PC (34:1) were increased. AGXT2L1 is an enzyme with strict substrate specificity for phosphoethanolamine, which is converted into ammonia in GW3965-treated hepatocytes and could explain the PE reduction. In summary, LXR activation by GW3965 targets PE biosynthesis and FA elongation/desaturation, which tends to decrease PE in relation to total PL levels, and remodelling of PC and PE molecular species. We identified the human AGXT2L1 gene as induced by LXR activation by both synthetic and endogenous agonist treatment. The increase in acetaldehyde-induced oxidative stress, and in the lipid species identified have the potential to enhance the inflammatory process and impair membrane function. Future studies should focus on inhibition of AGXT2L1 activity with the aim of reverting the steatosis induced by LXR activation.

The Nuclear Receptor-Co-repressor Complex in Control of Liver Metabolism and Disease.

Hepatocytes are the major cell-type in the liver responsible for the coordination of metabolism in response to multiple signaling inputs. Coordination occurs primarily at the level of gene expression via transcriptional networks composed of transcription factors, in particular nuclear receptors (NRs), and associated co-regulators, including chromatin-modifying complexes. Disturbance of these networks by genetic, environmental or nutritional factors can lead to metabolic dysregulation and has been linked to the progression of non-alcoholic fatty liver disease (NAFLD) toward steatohepatitis and even liver cancer. Since there are currently no approved therapies, major efforts are dedicated to identify the critical factors that can be employed for drug development. Amongst the identified factors with clinical significance are currently lipid-sensing NRs including PPARs, LXRs, and FXR. However, major obstacles of NR-targeting are the undesired side effects associated with the genome-wide NR activities in multiple cell-types. Thus, of particular interest are co-regulators that determine NR activities, context-selectivity, and associated chromatin states. Current research on the role of co-regulators in hepatocytes is still premature due to the large number of candidates, the limited number of available mouse models, and the technical challenges in studying their chromatin occupancy. As a result, how NR-co-regulator networks in hepatocytes are coordinated by extracellular signals, and how NR-pathway selectivity is achieved, remains currently poorly understood. We will here review a notable exception, namely a fundamental transcriptional co-repressor complex that during the past decade has become the probably most-studied and best-understood physiological relevant co-regulator in hepatocytes. This multiprotein complex contains the core subunits HDAC3, NCOR, SMRT, TBL1, TBLR1, and GPS2 and is referred to as the "NR-co-repressor complex." We will particularly discuss recent advances in characterizing hepatocyte-specific loss-of-function mouse models and in applying genome-wide sequencing approaches including ChIP-seq. Both have been instrumental to uncover the role of each of the subunits under physiological conditions and in disease models, but they also revealed insights into the NR target range and genomic mechanisms of action of the co-repressor complex. We will integrate a discussion of translational aspects about the role of the complex in NAFLD pathways and in particular about the hypothesis that patient-specific alterations of specific subunits may determine NAFLD susceptibility and the therapeutic outcomes of NR-directed treatments.

Hepatocyte-specific loss of GPS2 in mice reduces non-alcoholic steatohepatitis via activation of PPARα.

Obesity triggers the development of non-alcoholic fatty liver disease (NAFLD), which involves alterations of regulatory transcription networks and epigenomes in hepatocytes. Here we demonstrate that G protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor (NCOR) and histone deacetylase 3 (HDAC3) complex, has a central role in these alterations and accelerates the progression of NAFLD towards non-alcoholic steatohepatitis (NASH). Hepatocyte-specific Gps2 knockout in mice alleviates the development of diet-induced steatosis and fibrosis and causes activation of lipid catabolic genes. Integrative cistrome, epigenome and transcriptome analysis identifies the lipid-sensing peroxisome proliferator-activated receptor α (PPARα, NR1C1) as a direct GPS2 target. Liver gene expression data from human patients reveal that Gps2 expression positively correlates with a NASH/fibrosis gene signature. Collectively, our data suggest that the GPS2-PPARα partnership in hepatocytes coordinates the progression of NAFLD in mice and in humans and thus might be of therapeutic interest.

Nuclear receptor ligands induce TREM-1 expression on dendritic cells: analysis of their role in tumors.

Dendritic cells (DCs) initiate adaptive immune responses after their migration to secondary lymphoid organs. The LXR ligands/oxysterols and the RXR ligand 9-cis Retinoic Acid (9-cis RA) were shown to dampen DC migration to lymphoid organs through the inhibition of CCR7 expression. We performed transcriptomics of DCs undergoing maturation in the presence of the LXR ligand 22R-Hydroxycholesterol (22R-HC). The analysis highlighted more than 1500 genes modulated by 22R-HC treatment, including the triggering receptor expressed on myeloid cells (TREM)-1, which was found markedly up-regulated. We tested the effect of other nuclear receptor ligands (NRL) and we reported the induction of TREM-1 following RXR, RAR and VDR activation. From a functional point of view, triggering of TREM-1 induced by retinoids increased TNFα and IL-1ß release, suggesting an active role of NRL-activated TREM-1+ DCs in inflammation-driven diseases, including cancer. Consistently with this hypothesis we detected DCs expressing TREM-1 in pleural effusions and ascites of cancer patients, an observation validated by the induction of TREM-1, LXR and RAR target genes when monocyte-DCs were activated in the presence of tumor-conditioned fluids. Finally, we observed a better control of LLC tumor growth in Trem-1-/- bone marrow chimera mice as compared to wild type chimera mice. Future studies will be necessary to shed light on the mechanism of TREM-1 induction by distinct NRL, and to characterize the role of TREM-1+ DCs in tumor growth.

Potential Role of Thyroid Receptor ß Agonists in the Treatment of Hyperlipidemia.

Thyroid hormones have important effects on cellular development, growth, and metabolism and are necessary for the healthy function of almost all tissues. Hyperthyroid patients with excess thyroid hormone levels experience tachycardia, fatigue, muscle wasting, and osteoporosis. However, although high thyroid hormone levels have adverse effects, efforts have been made to harness the beneficial effects, such as reduced serum low-density lipoprotein (LDL) cholesterol levels, elevated basal metabolic rate, and weight loss. Thyroid hormones interact with nuclear thyroid hormone receptors (TRs), and cholesterol levels are reduced through TRß, whereas extrahepatic adverse actions are primarily connected to TRα. Thus, to develop a useful compound for clinical use, efforts have been focusing on developing compounds with isomer-specific functions based on the structure of thyroid hormones, i.e., thyromimetics that are liver and/or TRß specific. In this short review, we discuss the development of the early thyromimetics that enabled, through modern molecular techniques, the progress towards improved design of TRß-selective thyromimetics. We also address the early promise shown in human clinical trials and the current status of these drugs and other emerging compounds.

Transcriptional repression in macrophages-basic mechanisms and alterations in metabolic inflammatory diseases.

Macrophage differentiation and signal responses are coordinated by closely linked transcriptional and epigenomic mechanisms that trigger gene expression. In contrast to well-characterized transcriptional activation pathways in response to diverse metabolic and inflammatory signals, we just begin appreciating that transcriptional repression is equally important. Here, we will highlight macrophage pathways that are controlled by multifaceted repression events, along with a discussion of underlying regulatory mechanisms and components. We will particularly discuss pro- versus anti-inflammatory action of a fundamental corepressor complex, transcription factor cross-talk, repression at enhancers and during elongation, and diverse corepressor knockout mouse models. We will finally emphasize how alterations of macrophage repression pathways in humans contribute to, or even cause, metabolic inflammatory diseases such as obesity and type 2 diabetes.

Targeting liver X receptors in inflammation.

INTRODUCTION: The two oxysterol receptors, 'liver X receptors (LXRs)' LXRα and LXRß, are amongst the emerging newer drug targets within the nuclear receptor family and targeting LXRs represents novel strategies needed for prevention and treatment of diseases where current therapeutics is inadequate. AREAS COVERED: This review discusses the current understanding of LXR biology with an emphasis on the molecular aspects of LXR signalling establishing their potential as drug targets. Recent advances of their transcriptional mechanisms in inflammatory pathways and their physiological roles in inflammation and immunity are described. EXPERT OPINION: The new discoveries of LXR-regulated inflammatory pathways have ignited new promises for LXRs as drug targets. The broad physiological roles of LXRs involve a high risk of unwanted side effects. Recent insights into LXR biology of the brain indicate a highly important role in neuronal development and a clinical trial testing an LXR agonist reported adverse neurological side effects. This suggests that drug development must focus on limiting the range of LXR signalling - possibly achieved through subtype, tissue specific, promoter specific or pathway specific activation of LXRs where a successful candidate drug must be carefully studied for its effect in the central nervous system.

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 LXRß 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.

Metabolic nuclear receptor signaling and the inflammatory acute phase response.

The acute phase response (APR) classically refers to the rapid reprogramming of gene expression and metabolism in response to inflammatory cytokine signaling. As components of the innate immune system, hepatocyte-derived acute phase proteins (APPs) play a central role in restoring tissue homeostasis. Recently, an intriguing 'metaflammatory' facet of the APR became evident with chronically elevated APP levels being connected to metabolic syndrome disorders. The causality of these connections is unclear but could relate to adverse metabolic and inflammatory disturbances, particularly those affecting lipoprotein properties, cholesterol metabolism and atherogenesis. Here we review these aspects with an emphasis on the emerging importance of lipid-sensing nuclear receptors (LXRs, LRH-1, PPARs), in conjunction with anti-inflammatory transrepression pathways, as physiological and pharmacological relevant modulators of the APR.

GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRbeta 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.

GPS2 is required for cholesterol efflux by triggering histone demethylation, LXR recruitment, and coregulator assembly at the ABCG1 locus.

Transcriptional coregulators, rather than ligand signals, are suspected to confer context and pathway specificity to nuclear receptor signaling, but the identity of such specifying coregulators and the underlying molecular mechanisms remain largely enigmatic. Here we address this issue in metabolic oxysterol receptor LXR pathways and describe the selective requirement of GPS2 for ABCG1 cholesterol transporter gene transcription and cholesterol efflux from macrophages. We implicate GPS2 in facilitating LXR recruitment to an ABCG1-specific promoter/enhancer unit upon ligand activation and identify functional links to histone H3K9 demethylation. We further describe fundamental differences between ABCG1 and ABCA1 with regard to GPS2 in relation to other coregulators, which are likely to apply to additional LXR-regulated genes. Our work identifies a coregulator-dependent epigenetic mechanism governing the access of a nuclear receptor to communicating regulatory regions in the genome. The pathway and coregulator selectivity of this mechanism implies pharmacological possibilities for the development of selective LXR agonists.

RAP250 is a coactivator in the transforming growth factor beta signaling pathway that interacts with Smad2 and Smad3.

RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-beta (TGF-beta) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-beta target gene PAI-1 after stimulation by TGF-beta when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-beta and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-beta and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-beta signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-beta signaling pathways.

Molecular basis for repression of liver X receptor-mediated gene transcription by receptor-interacting protein 140.

Similarities in physiological roles of LXR (liver X receptors) and co-repressor RIP140 (receptor-interacting protein 140) in regulating energy homoeostasis and lipid and glucose metabolism suggest that the effects of LXR could at least partly be mediated by recruitment of the co-repressor RIP140. In the present study, we have elucidated the molecular basis for regulation of LXR transcriptional activity by RIP140. LXR is evenly localized in the nucleus and neither the N-terminal domain nor the LBD (ligand-binding domain) is necessary for nuclear localization. Both LXR subtypes, LXRalpha and LXRbeta, interact with RIP140 and co-localize in diffuse large nuclear domains. Interaction and co-localization are dependent on the LBD of the receptor. The C-terminal domain of RIP140 is sufficient for full repressive effect. None of the C-terminal NR (nuclear receptor)-boxes is required for the co-repressor activity, whereas the NR-box-like motif as well as additional elements in the C-terminal region are required for full repressive function. The C-terminal NR-box-like motif is necessary for interaction with LXRbeta, whereas additional elements are needed for strong interaction with LXRalpha. In conclusion, our results suggest that co-repression of LXR activity by RIP140 involves an atypical binding mode of RIP140 and a repression element in the RIP140 C-terminus.