ACTIVATION OF LXR/RXR PATHWAY AMELIORATES LIVER DISEASE IN ATP7B^−/− (WILSON DISEASE) MICE

Abstract

Wilson disease (WD) is a hepatoneurologic disorder caused by mutations in the copper-transporter ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurologic symptoms. We demonstrate that in Atp7b^−/− mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b^−/− mice identified disregulation of nuclear receptors (NR), especially the LXR/RXR heterodimer, as an important event in WD pathogenesis. Treating Atp7b^−/− mice with the LXR agonist T0901317 ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized and liver function and histology was improved. In conclusion, the results demonstrate the major role of an altered nuclear receptor function in the pathogenesis of WD; and suggest that modulation of nuclear receptor activity should be explored as a supplementary approach to improving liver function in WD.

Wilson Disease (WD) is a potentially fatal disease caused by mutations in the ATP-dependent copper transport protein, ATP7B. In hepatocytes, ATP7B facilitates copper delivery to the copper-dependent ferroxidase, ceruloplasmin, which undergoes functional maturation in a secretory pathway. ATP7B also maintains cytosolic copper at a non-toxic level by sequestering excess copper in vesicles for subsequent export into bile (1). WD causing mutations disrupt ATP7B function, resulting in accumulation of Cu in tissues, especially liver. The disease manifestations are highly variable, indicative of modifying factors, and pose significant challenges for diagnosis and treatment (1, 2). Copper chelation is the major and largely successful therapy for WD. However, frequent side effects (3), poor compliance, neurologic decompensation, and cost complicate therapy (2, 4). Development of additional or supplementary approaches requires a better understanding of the pathogenesis of WD. Atp7b^−/− mice are established model for studies of WD (5). These animals recapitulate the major manifestations of WD, including hepatic copper overload, loss of ceruloplasmin activity, elevated urine copper, and liver disease. In both mice and humans, the severity of pathology is not proportional to the amount of hepatic copper, further indicating a modifying influence of metabolic and/or environmental factors. A significant role for metabolism is also suggested by discordant clinical disease in monozygotic WD twins with different nutritional histories (6).

Our previous studies identified lipid metabolism, especially cholesterol biosynthesis, as the major metabolic pathway inhibited in response to hepatic copper accumulation in Atp7b^−/− mice prior to the onset of hepatitis (8). Similar findings in LEC rats (7) prompted human studies that described lower total cholesterol in WD patients compared to patients with comparable liver disease (8). Diminished levels/activity of HMG-CoA reductase is observed in all three species (7, 9). Here, we expand upon this work to identify the molecular basis of lipid dysregulation in WD. Our study provides direct evidence that abnormally reduced function of nuclear receptors involved in the reciprocal regulation of lipid metabolism and inflammatory response is the major event that triggers the onset and progression of liver pathology in WD. Furthermore, we demonstrate that upregulation of nuclear receptor function can ameliorate the disease even in the presence of high copper. These findings provide a foundation for future efforts to develop novel treatments to supplement chelation in WD.

Methods

Human studies

In accordance with Declaration of Helsinki and Institutional Review Board (U. Leipzig Reg. # 236-2006; U. Heidelberg Reg. # 346/2005) approved consent, WD patients underwent liver transplantation for acute or chronic liver failure. Control specimens were obtained from patients who underwent liver resections for other clinical reasons (Suppl. Tab. 1). Liver sections were snap frozen in liquid nitrogen and stored. Total RNA was isolated using TRIZOL reagent (Invitrogen, Grand Island, NY) followed by RNeasy cleanup procedure (Qiagen, Valencia, CA). The integrity of isolated RNA was verified by ethidium bromide staining and by optical densities (OD) ratio (OD260nm/280nm>1.8). A total of 16 liver RNA samples (8 biological replicates for control liver and 8 for WD patients) were examined for RNA integrity and concentration on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6.000 LabChip Kit (Agilent Technologies) according to the manufacturer’s instructions.

Affymetrix GeneChip analysis was conducted at the microarray core facility of the Interdisziplinäres Zentrum für klinische Forschung (IZKF), Leipzig (Faculty of Medicine, University of Leipzig). 2 μg of total RNA were used to prepare double-stranded cDNA (Superscript II, Life Technologies, Gaithersburg, MD) primed with oligo-dT containing an T7 RNA polymerase promoter site (Genset SA, Paris, France). cDNA was purified by phenol-chloroform extraction before in vitro transcription using the IVT labeling kit (Affymetrix, Santa Clara, CA, USA) to synthesize cRNA. After the in vitro transcription, unincorporated nucleotides were removed using the RNeasy kit (QIAGEN). The cRNA was fragmented and hybridized to two different (technical replicates) Human Genome U133 Plus 2.0 (Arrays Affymetrix). The washing and staining of the probe array was performed according to the manufacturer’s instructions. The array was scanned with a third generation Affymetrix GeneChipScanner 3000.

Image processing and analysis were performed using Affymetrix MAS 5.0 software. The resulting intensities and coordinate information were saved in a CEL file format and then subjected to global scaling with an average target intensity of 350 to allow for direct comparison of hybridization values from different targets. Scaled results for each sample were saved as CHP files and these data were used to evaluate overall chip performance. The analysis indicated that the parameters describing the quality of RNA, hybridization, and detection were all within acceptable range.

The associations between altered genes or pathways were evaluated using the Ingenuity Pathways Analysis software (Ingenuity® Systems, www.ingenuity.com). Affymetrix identifiers of the differentially expressed genes (the fold-change of 2.0 or higher) and their corresponding expression values were loaded into the software and mapped to its corresponding gene object (so-called focus genes) in the Ingenuity Pathways Knowledge Base. The significance of the associations between the data set and the canonical pathway and functional annotations was calculated in two ways. First, the number of genes from the data set mapping to a pathway was divided by the number of all known genes ascribed to the pathway. Second, the left-tailed Fischer’s exact test was used to calculate related p-values and distinguish those functional/pathway annotations which had more Focus Genes than expected by chance. The networks of the focus genes were algorithmically generated based on their connectivity.

Animal studies

ATP7b knockout (ATP7b ^−/−) and heterozygote (ATP7b ^+/−) mice were generated as previously prescribed (10). T0901317 (Cayman Chemical, Ann Arbor, MI) powder was mixed with Teklad 2018 powdered chow (Harlan, Madison WI) and fed to animals aged 6–7 weeks old at a dose of 50 mg/kg/day, thrice weekly. After 8 weeks, animals were sacrificed. The animals were housed at the Johns Hopkins University School of Medicine (JHUSOM) Animal Care Facility and protocols designed according to National Institutes of Health (USA) guidelines. Normal pellet chow was fed to the mice on days they were not receiving the drug. Animals were weighed weekly. At the time of sacrifice, trunk blood and livers were harvested. The Institutional Animal Care and Use Committee (IACUC) of JHUSOM approved the above experimental protocols.

Serum Biochemical Analysis

Animals were fasted overnight before sacrifice. Whole blood was collected from the aorta and vena cava in amber Microtainer tubes (Beckton Dickinson, Franklin Lakes, NJ), and serum was separated after centrifugation at 5000 rpm for 10 minutes. Liver biochemistries and lipid panels were performed in the Molecular and Comparative Pathobiology Core Laboratory of JHUSOM.

Quantitative RT-PCR

Sections of mouse liver were immersed in RNAlater, homogenized, and frozen at −80°C. RNA was isolated with TRIzol via the manufacturer’s instructions, then transcribed into cDNA using the SuperScript III first strand synthesis system (Invitrogen). RNA quantity was determined by an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). Samples were run in triplicate and performed on a 7900 HT machine (Applied Biosystems) and analyzed with the SDS 2.4.1 software. GAPDH served as the control to which gene of interest expression was normalized.

Quantitative RT-PCR for ABC1, HmGCoA1, CYP71a, FASN, LXRα (NR1H3), LXRβ (NR1H2), RXRα (NR2B2), SREBP1, FXR, TIMP1, COL1a, and GAPDH was performed with sequence-specific primers and probes using TaqMan gene expression assays (Applied Biosystems, Foster City, CA).

Immunoblotting

Antibodies were purchased from Santa Cruz Biotechnology, Inc (Dallas), Cell signaling (Beverly, MA), or Abcam (Cambridge, MA). Cytoplasmic and nuclear protein was extracted using the NE-PER^™ kit (Thermo Scientific). Immunoblotting for LXRα (SC-1202), LXRβ (SC-1203), RXR (SC-831), p38 MAPK (CS-8690S), P-p38 MAPK (CS-4511S), SAPK/JNK (CS-9252S), P-SAPK/JNK (CS-9251S), ERK 1/2 (CS-9102S), P-ERK 1/2 (CS-9101S), and GAPDH (ab8245) was performed as previously described (11). Densitometry was performed using Image J software (National Institutes of Health, Bethesda).

Histology

Sections of mouse liver were covered in OCT and snap frozen in liquid nitrogen. Sections were then cut and stained with hematoxylin and eosin. De-identified samples were analyzed for the presence of inflammation by an expert in veterinary pathology (D.H.). Inflammation was scored as follows: none, 0; mild, 1; moderate, 2; and severe, 3. Severely inflamed livers had karyomegaly, cytomegaly, binucleate hepatocytes, intranuclear vacuoles, multiple nuclei, and inflammatory cell infiltrates. Sirius red staining and subsequent morphometric analysis was performed as previously described (12).

Testing Copper binding to T0901317

0.5 mM Cu(I) stock solution was prepared by diluting CuCl₂ into 20 mM HEPES, pH 7.4 buffer with 2 mM tris(2-carboxyethyl)phosphine (TCEP). T0901317 stock solution was prepared in DMSO. For the BCS titration experiment, 0.5 mM Cu(I) was gradually added to 10 mM BCS in a 0.6 ml volume of 20 mM HEPES, pH 7.4 solution, to a final concentration of 8.3 mM. Coordination of Cu(I) to BCS was examined by UV-vis spectroscopy by monitoring the changes in absorption at 484 nm. For the T0901317 titration experiment, 0.5 mM Cu(I) was gradually added to 10 mM T0901317 in a 0.6 ml volume of DMSO solution, to a final concentration of 16.7 mM. Coordination of Cu(I) to T0901317 was examined by UV-vis spectroscopy by monitoring the absorption changes in the range from 300 to 800 nm.

Hepatic Copper measurements

Hepatic copper levels were measured by polarized atomic absorption spectroscopy as previously described (5). Briefly, 50–100 mg of liver was dissolved in 2ml of HNO₃ at 90°C. Copper levels were determined using a Hitachi Z-8279 spectrophotometer. Samples were compared to freshly prepared standards.

Hepatic Triglyceride measurements

The triglyceride colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) was used for measuring hepatic triglyceride content using the provided protocol. 120–160 mg of liver tissue was homogenized and samples were assayed in triplicate. Absorbance at 540 nm was detected on an Envision plate reader (Perkin-Elmer). Triglyceride concentrations were calculated using a standard curve, then normalized to the volume of homogenate and the mass of the liver tissue.

Hepatic Oxysterol measurements

Oxysterols were extracted and measured using the protocol described by McDonald et al (13). 1 mg of liver tissue was macerated, solubilized with methylene chloride and methanol, saponified with 10N KOH, and purified with hexane and aminopropyl SPE cartridges (Biotage, Uppsula, Sweden). Mass spectrometry was carried out using LC/MS on a triple quadrupole mass spectrometer (AB SCIEX 5500, Framingham, Massachusetts) in multiple reaction monitoring (MRM) mode. The HPLC column used for these analyses was 2×150 mm 2.6 mm particle size Kinetix column (Phenomenex Torrance, CA) at a flow rate of 2.5 ml/min. Quantitation was performed by establishing a linear relationship between measured amounts of the analytical standards combined with the internal standard cocktail. This regression line was applied to the measured peak areas for analytes in the extracted samples to determine measured quantities present in each sample. The numbers were normalized to the wet weight of the tissue extracted and reported as picograms of analyte/wet weight tissue.

Statistical Analysis

Data are presented as the mean and the standard error (SE) of the mean. The data were analyzed with the two-tailed student’s t-test, and p values less than 0.05 were considered significant.

Results

Lipid metabolism is abnormal in WD patients

Lipid metabolism in Atp7b^−/− mice is significantly affected by copper overload, even before liver disease is detected (9). The mRNA profiling and pathway analysis suggests that down-regulation of cholesterol biosynthesis is due to inhibition of signaling by nuclear receptors, especially the LXR/RXR heterodimer (Fig. 1A). (9) To examine relevance of these observations to human WD, we performed mRNA profiling of liver explants from 6 WD patients (2 samples did not pass quality control standards) using microarrays, and compared the profiles to those from 8 control individuals. All WD livers were at the advanced stage of disease (Suppl. Table 1), which produced changes in a large number of biological processes and pathways (Suppl. Tables 2, 3). The most significant altered biological processes were “metabolism” (824 genes), “positive and negative regulation of biologic process” (273 and 257 genes, respectively), “immune system process” (172 genes), “cell proliferation” (143 genes) and “biologic adhesion” (108 genes). Within the category of metabolism, “lipid metabolism” was the most significantly changed (220 genes), with a highly significant Z-score of 15.54. The primary change was down-regulation (165 genes). The pathways most affected were LXR/RXR activation (downregulated) and IL-1β inactivation of RXR (upregulated) (Fig. 1B). Changes in lipid and carbohydrate metabolism represent the largest percentage of metabolic changes (32.76%) with lipid metabolism responsible for most of these changes (24%) (Fig. 1C) The most significant change was down-regulation of signaling mediated by the FXR/RXR or LXR/RXR nuclear receptors (47/116 genes affected, p=2.28×10⁻²², Fig 1B and C and Suppl. Tables 2 and 3).

Fig. 1.

Taken together, the in silico analysis of gene expression indicated that LXR/RXR mediated regulation of lipid metabolism was the most affected pathway in patients with WD and Atp7b^−/− mice, and therefore a promising target for further investigation.

The level of RXR nuclear receptor and LXR/RXR targets are decreased in Atp7b^−/− mice

The functional status of LXR/RXR pair was evaluated by measuring expression levels of LXR/RXR targets - fatty acid synthase and HMG-CoA reductase. The mRNA levels for both genes were significantly down-regulated in Atp7b^−/− livers consistent with lower LXR/RXR activity (Fig. 2A). The apparently diminished LXR/RXR function could be either due to decreased levels of endogenous activating ligands, or due to lower expression of these receptors. Oxysterols (OHC) are potent activators of LXR. Mass-spectrometry analysis of OHC profiles demonstrated that the mean levels of LXR ligands 24S-OHC and 22R-OHC were lower in Atp7b^−/− mice compared to control, however the decrease was not statistically significant. Statistically significant decreases were observed for 7α, β-OHC (produced by the FXR target Cyp7a1) and 5,6 β-epoxycholesterol (5,6β-EC). By comparison, the levels of 4β-hydroxycholesterol were higher in Atp7b^−/− mice compared to control (Fig. 2B); this OHC is a marker of activation of CYP3A4/5, which is regulated primarily by constitutive androstane receptor (CAR)-mediated signaling, rather than LXR(14).

Fig. 2.

Since the decrease in LXR ligands was modest, we also examined abundance of LXRα, LXRβ and RXR. LXRα mRNA (NR1H3) was decreased in the Atp7b^−/− liver, whereas protein levels were not changed significantly. There was no difference in LXRβ (NR1H2) or RXRβ (NR2B2) mRNA (Fig. 2A) and protein levels of LXRβ were only slightly decreased. In contrast, RXR was significantly less abundant in both the cytoplasmic and nuclear fractions from the Atb7b^−/− liver (Figure 2C and Suppl. Figure 1). Thus, lower expression of LXR/RXR-regulated genes could be due to decreased RXR and a reduced number of functional LXR/RXR dimers. Oxidative stress is known to stimulate the phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK, which represses RXR expression and signaling (15, 16). However, we did not find evidence of increased phosphorylation of these proteins (Suppl. Fig 2A–F).

Treatment with LXR agonist T0901317 does not prevent copper accumulation in the liver

Activation of LXR/RXR upregulates lipid metabolism and inhibits inflammatory response (17). We hypothesized that reductions in LXR/RXR function may be responsible for the inhibition of cholesterol biosynthesis and increased hepatic inflammation observed in WD patients and Atp7b^−/− mice, and play an important role in the development of liver pathology in WD. If true, activation of the LXR/RXR pathway may prevent hepatitis and improve liver function. To test this hypothesis, we examined the effect of an LXR agonist (T0901317) on copper levels, lipid metabolism, liver morphology and function in Atp7b^−/− mice (KO). The histologically and biochemically normal heterozygote (HET) mice were used as controls.

The HET animals that received the drug were significantly heavier than the age-matched controls (26.43±1.35g vs. 22.53±0.72g, p<0.05, Suppl. Fig. 3A), whereas there was neither a difference in the body weights (24.03±0.91g vs. 24.67±1.54) nor the liver weights between the treated and untreated KO animals (Suppl. Fig. 3B.). Copper overload is the initial metabolic trigger for development of liver pathology in WD and it was important to determine that the drug acts down-stream of copper by activating lipid metabolism and not by diminishing copper levels in tissue. At the end of the experiment, copper levels in the KO mice were increased 38-fold compared to HET control. Similar increase was observed in drug-treated Atp7b^−/− mice (Fig. 3A), indicating that T0901317 did not prevent copper accumulation. We then tested directly whether T0901317 can bind Cu(I) and observed no binding (Suppl. Fig 4), further arguing against a possibility that the LXR agonist acts as a copper chelator.

Fig. 3.

T0901317 normalizes lipid metabolism in Atp7b^−/− mice by upregulating expression of a subset of the LXR target genes

LXR is a transcription factor for multiple genes that regulate cholesterol synthesis, transport and metabolism. In the untreated Atp7b^−/− mice, in addition to fatty acid synthase (FASN), and 3-hydroxyl-3-methylglutaryl-CoA synthase 1 (HMGCS1), the expression of NR1H4, and cytochrome P450 family 7 subfamily A polypeptide 1 (CYP7A1) was significantly lower compared to the untreated controls, whereas transcripts for ATP-binding cassette 1 (ABC1) and Sterol regulatory element-binding protein 1 (SREBP1) were not significantly different. Treatment with T0901317 significantly increased the FASN expression in both Atp7b^+/− mice and Atp7b^−/− mice. SREBP1 mRNA was increased in the ATP7b^+/− mice after the treatment, but there was no change in the ATP7b^−/− mice. In both KO and HET mice, the treatment did not change mRNA levels for NR1H2, NR1H3, NR1H4, ABC1, HMGCS1, or CYP7A1 (Suppl. Fig. 5).

The mixed mRNA response to T0901317 indicated that only a subset of predicted targets of LXR/RXR pathway was stimulated by the drug. RXR dimerizes with other nuclear receptors (Fig 1a), so the observed partial correction of transcriptional changes could be due to contributions from other RXR-dependent pathways. Treatment with T0901317 did not change the levels of LXRα, LXRβ, or RXR in Atp7b^+/− mice or Atp7b^−/− mice (Suppl. Fig 1B.) Given this partial response, we directly tested whether treatment with T0901317 reversed changes in lipid metabolism. The livers of both the drug treated HET and KO mice showed a statistically significant increase in triglycerides compared to the respective untreated controls (Fig. 3B), which was expected for treatment with T0901317 (18). Plasma lipid analysis revealed that total cholesterol, low density lipoprotein (LDL), and high density lipoprotein (HDL) levels were all significantly increased in the sera of the drug treated Atp7b^−/− mice compared to untreated Atp7b^−/− mice (p<0.001, p<0.001, and p<0.005, respectively). Importantly, although the total cholesterol, LDL, and HDL more than doubled in the treated Atp7b^−/− mice compared to the untreated mice, these resulting levels were similar to treated and untreated heterozygote mice (Fig. 3C). Collectively, the increases in hepatic triglycerides and serum lipids in the treated mice are consistent with activation of LXR/RXR signaling.

Treatment with T0901317 decreases inflammation and liver fibrosis in Atp7b^−/− liver

Chronic inflammation underlies the development of liver fibrosis and ultimately, cirrhosis. Inactivation of LXR/RXR is expected to relieve inhibition of expression of genes associated with the NF-KappaB inflammatory cascade(19), whereas treatment with LXR/RXR agonist would facilitate inhibition. Indeed, untreated Atp7b^−/− mice had a 10-fold increase in TNFα, a 2-fold increase IL-1β, and a 25-fold increase in iNOs expression when compared to the controls. Treatment with T0901317 significantly reduced the expression of these genes in the Atp7b^−/− mice (Fig. 4A). There was no effect of drug treatment in the heterozygote animals.

Fig. 4.

Inflammatory cytokines TNFα and IL-1β activate hepatic stellate cells (20) to produce collagen 1a, leading to fibrosis. The Atp7b^−/− mice have a more than 25-fold increase of Col1a mRNA compared to the control mice. This increase in expression of collagen was completely abolished by treatment with T0901317 (Fig. 4B). Similarly, Tissue inhibitor of metalloproteinase 1 (Timp-1), a marker of inflammatory/fibrotic liver disease (20), was 20-fold higher in the untreated Atp7b^−/− mice and similar to control following treatment with the drug (Fig. 4B).

LXR agonist improves liver function

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are enzymes elevated in the sera during liver inflammation and injury. In Atp7b^−/− mice, the AST levels were 626±142 U/L and ALT was 666±85 U/L. Treatment with T0901317 significantly reduced the liver transaminases compared to untreated Atp7b^−/− mice (p<0.05). The mean AST was reduced to 222±64 U/L and ALT to 285±115 U/L. The liver enzymes (particularly AST) in the drug treated KO mice were similar to drug-treated heterozygote mice (Fig. 4C). Compared to non-treated Het mice, AST and ALT were higher in the drug-treated Het mice suggesting that prolonged administration of T0901317 may result in hepatotoxicity (Fig. 4C). Liver function was further assessed by serum bilirubin and albumin levels. In treated Atp7b^−/− mice the total bilirubin was significantly lower than in the untreated Atb7b^−/− mice (0.85±0.22 mg/dL vs. 1.96±0.39 mg/dL, p<0.05). Similarly, treatment improved the albumin levels to 2.93±0.19 mg/dL vs. 2.27±0.09 mg/dL in the untreated, knockout mice (p<0.05). (Fig. 4D).

Liver histology was examined by light microscopy and confirmed significant improvements in the drug treated KO mice. Untreated KO mice livers have ballooning hepatocytes, intracytoplasmic vacuoles, karyomegaly, multiple nuclei, and inflammatory cell infiltrates. These pathologic features disappeared in 5/7 treated KO mice (Fig. 5A and 5B). Although aminotransferases were increased in the Het mice compared to control, the treatment had no effect on the liver histology in 7/8 heterozygote mice (Fig 5B and Suppl. Fig. 6).

Fig. 5.

Morphometric analysis of Sirius red stained liver sections revealed a more than 50% (p<0.001) decrease in liver fibrosis in the treated KO mice, compared to control KO mice. There was a small yet significant increase in Sirius red staining in the treated Het mice when compared to control Het mice. Untreated KO mice had a 4-fold increase in liver fibrosis compared to untreated Het mice. (Fig. 5C, D)

Altogether, our findings are summarized in Fig. 6. The results suggest copper accumulation caused by inactivation of Atp7b is associated with inhibition of nuclear receptor signaling, especially, but not exclusively, the LXR/RXR heterodimer. We propose that the combined effects of reduced LXR/RXR activity in hepatocytes, stellate cells, and Kupffer cells all contribute to the pathogenesis of WD. Activation of this pathway with an endogenous agonist improved liver inflammation, histology, and liver function but did not alter hepatic copper.

Fig. 6.

Discussion

We demonstrate that the genetic program governing lipid metabolism and cholesterol biosynthesis is down-regulated in patients with Wilson disease and in Atp7b knockout mice. The change in transcriptional profiles is associated with decreased levels of cholesterol and triglycerides in serum and increased liver inflammation. In silico analysis linking gene expression to signaling pathways and metabolic processes pointed to inhibition of nuclear receptors in both mice and man as a primary cause of the observed changes. Consistent with this prediction, stimulation of nuclear receptor activity using the LXR agonist, T0901317, abrogated the major negative consequences of copper overload and improved liver morphology and function. Significantly, in the presence of the drug, the liver function was maintained (or had less decompensation) for a prolonged period of time despite elevated hepatic copper. We propose that in WD, accumulating copper inhibits nuclear receptor signaling, which is a critical mediator of liver injury in WD. This represents a novel mechanism in the pathogenesis of WD.

Copper chelation is an established and by in large successful approach to decreasing copper toxicity in WD. Life-long treatment using copper chelators improves liver function, but also has a wide range of side effects. Reaching a delicate copper balance in different tissues is difficult and prolonged chelation frequently produces neurologic decompensation and has other unintended consequences. Targeting pathways downstream of copper offers an alternative strategy for improving liver function in WD, especially under circumstances when copper chelators are ineffective or poorly tolerated. At the current stage, our data represent “a proof of concept” that stimulating nuclear receptor function can greatly diminish pathologic changes in WD liver. In this study, we initiated treatment with the drug when copper was high, lipid profile was altered, and AST/ALT were elevated, but the structural morphology of liver was still preserved, and we obtained very promising results. Whether similar treatment will be equally effective if started at a more advanced stage of the disease needs to be determined. It is likely that treatment combining mild copper chelation and activation of nuclear receptor signaling could be most effective.

Inflammation, lipid dis-homeostasis eventually leading to steatosis, and fibrosis are commonly observed in WD. Many of the genes regulating these processes are under the control of nuclear receptors, including the LXR/RXR heterodimer. The LXRα and LXRβ levels were unchanged in the KO mice, whereas RXR levels were lower in the KO mice (see also Muchenditsi et al). A lower abundance of RXR is likely to decrease transcriptional activity of the LXR/RXR heterodimer, although this remains to be formally demonstrated. Interestingly, IL-1β expression transiently stimulates SAPK/JNK resulting in RXR export from the nucleus, followed by proteosomal degradation(21). IL-1β is elevated in the WD mice, suggesting a possible explanation for diminished protein levels of RXR. Another factor potentially contributing to lower abandance/activity of RXR are levels of endogenous ligands. Oxysterols regulate activity of the LXR/RXR dimer, and the oxysterol profile differed significantly between the control and knockout animals. In particular, 7α,β-OHC and 5,6β-EC levels were significantly lower in the knockout animals compared to healthy controls, whereas 4β-OHC was increased in the ATP7b deficient mice. Treatment with the drug did not reverse changes in oxysterol levels (not shown), indicating that that the cause for oxysterol misbalance was upstream (or produced independently) of LXR/RXR signaling. Such causes may involve copper dependent, non-enzymatic oxidation and/or inhibition of activity of enzymes involved in oxysterol metabolism.

The LXR agonist used in this study had an uneven effect on gene expression. Genes commonly associated with liver fibrosis (COL1a and TIMP1) and liver inflammation (TNFα, IL-1β, and iNOS) were dramatically down-regulated by the treatment in knockout animals, suggesting primary control by LXR. At the same time, only a sub-set of genes involved in lipid metabolism responded to treatment with the drug. This result indicates that the decrease in RXR may have a negative effect on the function of several receptors involved in lipid metabolism with which RXR dimerizes, such as FXR, PPAR, and RAR. Despite partial recovery of mRNA levels, serum lipids in the treated mice increased to levels similar to those found in control mice. In addition, hepatic triglyceride content was increased in the treated mice, strongly suggesting the recovery of LXR signaling in the treated Atb7b^−/− mice.

Our findings of reduced Col1A, and TIMP-1 in treated Atb7b^−/− mice corroborate previous studies that indicate LXR activation in hepatic stellate cells is a critical determinant of liver inflammation and fibrosis (Fig 6.). LXRα/β^−/− mice have an exaggerated inflammatory and fibrotic response to classic experimental hepatic insults. Furthermore, bone marrow transplants between wild type and LXRα/β^−/− mice had no effect on the severity of fibrosis, suggesting the LXR signaling in hepatic stellate cells and/or hepatocytes is critical for the development of hepatic fibrosis (28). Future studies are needed to test the effects of ATP7b deletion, copper overload, and NR function in specific cell types in the liver.

Treatment of hepatoblastoma cells (HepG2) with copper induces apoptosis that is mediated by an increase in acid sphingomeylinase (ASMase) activity and production of ceramide (29). ASMase activity is increased by TNFα and ASMase deficiency renders cultured hepatocytes resistant to TNFα mediated apoptosis (30). We observed high levels of TNFα mRNA in liver extracts from the Atp7b KO mice, and the LXR agonist dramatically reduced the TNFα. Analysis of sphingolipids in one KO animal and age-matched control showed increased levels of ceramide, although separate study is needed to determine significance of this pathway in mice and humans. Elevated levels of serum TNFα (31) and IL-1β (32) are found in patients with WD (as in our mouse model).

The beneficial effects of T0901317 in WD mice are tempered by the increase in liver enzymes and fibrosis in the treated, control mice. Review of the histology indicates that increased hepatic steatosis is the most likely cause of the liver injury. In our study we treated the mice for 2 months, and although we found increased levels of AST and ALT, there was no significant increase in bilirubin or decrease in albumin. Moreover, there were no increases or changes in inflammatory genes (TNFalpha, Il-1Beta, NOS). Newer generation LXR agonists in development do not cause hepatic lipogenesis (35, 36). In addition, there are several concomitant treatments that can suppress T0901317 induced steatosis such as Resveratrol (37), Ursodeoxycholic acid (38), and n-3 fatty acids (39). Thus, future modifications or adjuvant therapies may diminish the observed toxic effects.

In conclusion, treatment with an LXR agonist improved liver histology, liver enzymes and liver function in a mouse model of WD. The improvements were associated with a decrease in genetic markers of liver fibrosis and inflammatory cytokines. There was no change in hepatic copper. These findings suggest alterations in nuclear receptor mediated lipid metabolism and inflammation may contribute to the pathogenesis of liver disease in WD, and that alternative therapies targeting these receptors are possible.

Abbreviations

WD

Wilson disease

ATP7B

ATPase Cu⁺⁺ transporting beta polypeptide

HMG-CoA

3-hydroxy-3-methylgultaryl-coenzyme A

LEC

Long-Evans Cinnamon rat

ABC1

ATP binding cassette 1

CYP7a1

cytochrome p450 family 7 subfamily A polypeptide 1

FASN

fatty acid synthase

SREBP1

sterol regulatory element-binding transcription factor 1

LXR

liver X receptor

RXR

retinoid X receptor

FXR

Farnesoid X receptor

Timp-1

tissue inhibitor of metalloproteinase 1

COL1a

collagen 1 alpha

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

p38-MAPK

p38 mitogen activated kinase

P

phosphylated

SAPK/JNK

stress activated kinase/c-Jun N-terminal kinase

ERK

extracellular signal-related kinase

OCT

optimal cutting temperature compound

HNO₃

nitric acid

KOH

potassium hydroxide

IL-1β

interleukin -1beta

TNFα

tumor necrosis factor alpha

iNOs

inducible nitric oxide synthase

OHC

hydroxysterol

EC

epoxycholesterol

KO

knock out

HET

heterozygote

CAR

constitutive androsterone receptor