Common Transcriptional Program of Liver Fibrosis in Mouse Genetic Models and Humans.

Multifactorial metabolic diseases, such as non-alcoholic fatty liver disease, are a major burden to modern societies, and frequently present with no clearly defined molecular biomarkers. Herein we used system medicine approaches to decipher signatures of liver fibrosis in mouse models with malfunction in genes from unrelated biological pathways: cholesterol synthesis-Cyp51, notch signaling-Rbpj, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling-Ikbkg, and unknown lysosomal pathway-Glmp. Enrichment analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome and TRANScription FACtor (TRANSFAC) databases complemented with genome-scale metabolic modeling revealed fibrotic signatures highly similar to liver pathologies in humans. The diverse genetic models of liver fibrosis exposed a common transcriptional program with activated estrogen receptor alpha (ERα) signaling, and a network of interactions between regulators of lipid metabolism and transcription factors from cancer pathways and the immune system. The novel hallmarks of fibrosis are downregulated lipid pathways, including fatty acid, bile acid, and steroid hormone metabolism. Moreover, distinct metabolic subtypes of liver fibrosis were proposed, supported by unique enrichment of transcription factors based on the type of insult, disease stage, or potentially, also sex. The discovered novel features of multifactorial liver fibrotic pathologies could aid also in improved stratification of other fibrosis related pathologies.

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis.

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.

Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells.

BACKGROUND AND AIMS: Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs). METHODS: Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression. RESULTS: Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [125I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [125I]-VEGF-A for at least 2 hours. Uptake of [125I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent internalization, whereas inhibition of cysteine proteases by leupeptin inhibited degradation without affecting the uptake of [125I]-VEGF-A, suggesting that it is degraded following transport to lysosomes. Incubation of LSECs in the continued presence of a saturating concentration of unlabeled VEGF-A at 37°C was associated with a loss of as much as 75% of the total VEGFR2 within 30 min as shown by Western blot analysis, whereas there was no appreciable decrease in protein levels for VEGFR1 after 120 min incubation, suggesting that VEGF-A stimulation downregulates VEGFR2, but not VEGFR1, in LSECs. This possibility was supported by the observation that a hexapeptide that specifically blocks VEGF-A binding to VEGFR1 caused a marked reduction in the uptake of [125I]-VEGF-A, whereas a control peptide had no effect. Finally, live cell imaging studies using a fluorescently labeled anti-VEGFR2 antibody showed that VEGFR2 was transported via early and late endosomes to reach endolysosomes where degradation of the VEGFR2 takes place. CONCLUSION: Our studies suggest that, subsequent to VEGF-A binding and internalization, the unoccupied VEGFR1 may recycle to the cell surface allowing its reutilization, whereas the majority of the internalized VEGFR2 is targeted for degradation.

The lysosomal transporter MFSD1 is essential for liver homeostasis and critically depends on its accessory subunit GLMP.

Lysosomes are major sites for intracellular, acidic hydrolase-mediated proteolysis and cellular degradation. The export of low-molecular-weight catabolic end-products is facilitated by polytopic transmembrane proteins mediating secondary active or passive transport. A number of these lysosomal transporters, however, remain enigmatic. We present a detailed analysis of MFSD1, a hitherto uncharacterized lysosomal family member of the major facilitator superfamily. MFSD1 is not N-glycosylated. It contains a dileucine-based sorting motif needed for its transport to lysosomes. Mfsd1 knockout mice develop splenomegaly and severe liver disease. Proteomics of isolated lysosomes from Mfsd1 knockout mice revealed GLMP as a critical accessory subunit for MFSD1. MFSD1 and GLMP physically interact. GLMP is essential for the maintenance of normal levels of MFSD1 in lysosomes and vice versa. Glmp knockout mice mimic the phenotype of Mfsd1 knockout mice. Our data reveal a tightly linked MFSD1/GLMP lysosomal membrane protein transporter complex.

ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines.

BACKGROUND: Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. RESULTS: By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. CONCLUSION: Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR.

Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator.

BACKGROUND: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1. RESULTS: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element. In transiently transfected Drosophila Schneider S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter. CONCLUSION: We propose that NCU-G1 is a dual-function protein capable of functioning as a transcription factor as well as a nuclear receptor co-activator.

Age-dependent development of liver fibrosis in Glmp (gt/gt) mice.

BACKGROUND: Mice lacking glycosylated lysosomal membrane protein (Glmp (gt/gt) mice) have liver fibrosis as the predominant phenotype due to chronic liver injury. The Glmp (gt/gt) mice grow and reproduce at the same rate as their wild-type siblings. Life expectancy is around 18 months. METHODS: Wild-type and Glmp (gt/gt) mice were studied between 1 week and 18 months of age. Livers were analyzed using histological, immunohistochemical, biochemical, and qPCR analyses. RESULTS: It was shown that Glmp (gt/gt) mice were not born with liver injury; however, it appeared shortly after birth as indicated by excess collagen expression, deposition of fibrous collagen in the periportal areas, and increased levels of hydroxyproline in Glmp (gt/gt) liver. Liver functional tests indicated a chronic, mild liver injury. Markers of inflammation, fibrosis, apoptosis, and modulation of extracellular matrix increased from an early age, peaking around 4 months of age and followed by attenuation of these signals. To compensate for loss of hepatocytes, the oval cell compartment was activated, with the highest activity of the oval cells detected at 3 months of age, suggesting insufficient hepatocyte proliferation in Glmp (gt/gt) mice around this age. Although constant proliferation of hepatocytes and oval cells maintained adequate hepatic function in Glmp (gt/gt) mice, it also resulted in a higher frequency of liver tumors in older animals. CONCLUSIONS: The Glmp (gt/gt) mouse is proposed as a model for slowly progressing liver fibrosis and possibly as a model for a yet undescribed human lysosomal disorder.

Increased glucose utilization and decreased fatty acid metabolism in myotubes from Glmp(gt/gt) mice.

Glycosylated lysosomal membrane protein (GLMP) has been reported to enhance the expression from a peroxisome proliferator-activated receptor alpha (PPARα) responsive promoter, but also to be an integral lysosomal membrane protein. Using myotubes established from wild-type and Glmp(gt/gt) mice, the importance of GLMP in skeletal muscle was examined. Glmp(gt/gt) myotubes expressed a more glycolytic phenotype than wild-type myotubes. Myotubes from Glmp(gt/gt) mice metabolized glucose faster and had a larger pool of intracellular glycogen, while oleic acid uptake, storage and oxidation were significantly reduced. Gene expression analyses indicated lower expression of three PPAR-isoforms, a co-regulator of PPAR (PGC1α) and several genes important for lipid metabolism in Glmp(gt/gt) myotubes. However, ablation of GLMP did not seem to substantially impair the response to PPAR agonists. In conclusion, myotubes established from Glmp(gt/gt) mice were more glycolytic than myotubes from wild-type animals, in spite of no differences in muscle fiber types in vivo.

Akt/PTEN signaling mediates estrogen-dependent proliferation of primordial germ cells in vitro.

Testicular tumors in humans are reported to be significantly increasing in incidence. Embryo exposure to environmental estrogens has been proposed as one of the possible underlying causes. In mice, genetic, immunological, and experimental evidence suggest that germ cell testicular tumors may derive from primordial germ cells (PGCs), the embryonic precursors of gametes. Here we show that relatively high concentrations of estrogens stimulate mouse PGC growth in vitro through the somatic cells of the gonadal ridges. Moreover, we found that estrogens stimulate the transcription of the Steel gene and the production of c-Kit ligand in gonadal somatic cells, and that this growth factor is likely to be responsible for the observed stimulation of PGC growth via an Akt/PTEN pathway. Finally, we show that estrogen stimulation of gonadal somatic cells in culture, in combination with PTEN down-regulation in PGCs and the presence of leukemia inhibitory factor in the culture medium, result in high frequency of PGC transformation in tumorigenic cells. Based on these results, we present a novel experimental in vitro model for tumorigenic germ cell transformation and identify molecular pathways likely involved in development of germ cell tumors after estrogen exposure.

Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver.

Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp gt/gt mice (formerly known as Ncu-g1gt/gt mice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.

Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells.

Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1(gt/gt) mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1(gt/gt) liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1(gt/gt) Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1(gt/gt) mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.

Bile acids reduce SR-BI expression in hepatocytes by a pathway involving FXR/RXR, SHP, and LRH-1.

Hepatic SR-BI mediates uptake of circulating cholesterol into liver hepatocytes where a part of the cholesterol is metabolised to bile acids. In the hepatocytes, bile acids reduce their own synthesis by a negative feedback loop to prevent toxic high levels of bile acids. Bile acid-activated FXR/RXR represses expression of CYP7A1, the rate-limiting enzyme during bile acid synthesis, by inducing the expression of SHP, which inhibits LXR/RXR and LRH-1-transactivation of CYP7A1. The present paper presents data indicating that CDCA suppresses SR-BI expression by the same pathway. As previously reported, LRH-1 induces SR-BI promoter activity. Here we show that CDCA or over-expression of SHP inhibit this transactivation. No FXR-response element was identified in the bile acid-responsive region of the SR-BI promoter (-1200bp/-937bp). However, a binding site for LRH-1 was characterised and shown to specifically bind LRH-1. The present study shows that also the SR-BI-mediated supply of cholesterol, the substrate for bile acid synthesis, is feedback regulated by bile acids.

Glucocorticoid response and promoter occupancy of the mouse LXRalpha gene.

The liver X receptors alpha and beta (LXRalpha and LXRbeta) are members of the nuclear receptor superfamily of proteins which are highly expressed in metabolically active tissues. They regulate gene expression of critical genes involved in cholesterol catabolism and transport, lipid and triglyceride biosynthesis, and carbohydrate metabolism in response to distinct oxysterol intermediates in the cholesterol metabolic pathway. Several LXR target genes have been identified, but there is limited information on how expression of the LXRs themselves is controlled. In this study we have characterized the upstream flanking region of the mouse LXRalpha gene. Transient transfections show that the LXRalpha promoter is able to drive transcription of a luciferase reporter gene, however, the transcriptional potential of the promoter in the cell lines used was low. The -2143 to -1513 region of the promoter mediates repression of reporter gene activity in all cells analyzed and multiple DNA-protein interactions were detected in this region by DNase I footprinting. The Zta, Ets, and Hes1 transcription factors were all shown to mediate alterations in reporter gene activity driven by LXRalpha promoter deletion constructs. These factors have been linked to cell cycle and differentiation processes suggesting that expression of LXRalpha might be under control of signalling mechanisms regulating cell proliferation. Several putative binding sites of the glucocorticoid receptor (GR) were identified in the LXRalpha promoter and transient cotransfections of the GR and LXRalpha promoter deletion constructs induced reporter gene activity. Addition of dexamethasone, a GR agonist, abolished this effect suggesting cross talk between GR and LXR signalling.

Oxysterol-activated LXRalpha/RXR induces hSR-BI-promoter activity in hepatoma cells and preadipocytes.

SR-BI mediates exchange of cholesterol between HDL and cells, and is a crucial factor in the transport of excessive cellular cholesterol from extrahepatic tissues to the liver ("reverse cholesterol transport") and, therefore, also for cholesterol homeostasis. Hepatic SR-BI mediates transfer of HDL-cholesterol to the hepatocytes where cholesterol may be metabolised to bile acids. LXR and SREBP are key factors in the regulation of cholesterol metabolism. The purpose of the present study was to determine whether these transcription factors are involved in the regulation of SR-BI. Here we show that LXRalpha/RXR and LXRbeta/RXR induce SR-BI transcription in human and murine hepatoma cell lines, and in 3T3-L1 preadipocytes independently of SREBP-1. The LXR/RXR response was mapped within -1,200 to -937 of the promoter region. Gel mobility shift analysis confirmed that the putative LXR response element bound LXRalpha/RXR and LXRbeta/RXR heterodimers.

Different regulation of the LXRalpha promoter activity by isoforms of CCAAT/enhancer-binding proteins.

LXRs have recently been shown to regulate key enzymes in cholesterol degradation, reverse transport of cholesterol from peripheral cells, cholesterol uptake and lipogenesis. The LXRalpha promoter was thus studied to investigate if LXRalpha gene expression is under the regulation of transcription factors involved in adipogenesis. We report that the C/EBP transcription factor interacts with the promoter of the LXRalpha gene. In in vitro footprinting experiments, protein extracts from several tissues gave footprints covering a putative C/EBP recognition site. Transfection experiments and EMSA showed a direct effect of these transcription factors on the LXRalpha promoter. C/EBPalpha upregulated expression of the reporter gene in an NIH 3T3-L1 preadipocyte cell line, while C/EBPbeta and C/EBPdelta had no effect. In liver hepatoma Fao II and Cos-7 kidney cells, both C/EBPalpha and C/EBPbeta downregulated expression of the reporter gene while C/EBPdelta induced activity, indicating that the functional consequences of C/EBP isoform interactions with the LXRalpha promoter are dependent on the cellular context. Monitoring of the LXR mRNA levels during adipose tissue differentiation showed that LXRbeta is constitutively expressed during the entire differentiation process while LXRalpha is induced upon addition of differentiation mix.