IFN-γ inhibits liver progenitor cell proliferation in HBV-infected patients and in 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-fed mice.

BACKGROUND & AIMS: Proliferation of liver progenitor cells (LPCs) is associated with inflammation and fibrosis in chronic liver diseases. However, how inflammation and fibrosis affect LPCs remains obscure. METHODS: We examined the role of interferon (IFN)-γ, an important pro-inflammatory and anti-fibrotic cytokine, in LPC expansion in HBV-infected patients and in mice challenged with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)- or choline-deficient, ethionine-supplemented (CDE) diet as well as in primary LPCs and LPC cell line. RESULTS: The CK19 staining scores correlated with inflammation and fibrosis grades in the livers from 110 HBV-infected patients. Nine-month IFN-γ treatment decreased LPC numbers, inflammation, and fibrosis in these HBV-infected patients. Similarly, a two-week IFN-γ treatment also decreased LPC activation in DDC-treated mice. Disruption of IFN-γ or its signaling components (e.g., IFNGR, STAT1, and IRF-1) increased LPC proliferation and liver fibrosis in DDC-fed mice. In contrast, deletion of IFN-γ did not increase, but rather slightly reduced LPC proliferation in CDE-fed mice. In vitro, IFN-γ attenuated proliferation of the LPC cell line BMOL and of primary LPCs from wild type mice, but not STAT1(-/-) or IRF-1(-/-) mice. Furthermore, co-culture assays suggest that IFN-γ can indirectly promote LPC proliferation via the activation of macrophages but attenuate it via the inhibition of hepatic stellate cells. CONCLUSIONS: IFN-γ inhibits LPC expansion via the direct inhibition of LPC proliferation and indirect attenuation of liver fibrosis in the DDC model, but it may also enhance LPC expansion via the promotion of inflammation in the CDE model; thereby playing dual roles in regulating LPC proliferation in vivo.

Hepatocellular carcinoma as extracolonic manifestation of Lynch syndrome indicates SEC63 as potential target gene in hepatocarcinogenesis.

OBJECTIVE: Lynch syndrome is a cancer predisposition syndrome caused by germline mutations in DNA mismatch repair (MMR) genes with microsatellite instability (MSI) as its molecular hallmark. Hepatocellular carcinoma (HCC) has not been considered part of the tumor spectrum. The aim was to provide a detailed molecular characterization of an HCC associated with Lynch Syndrome (Muir-Torre variant). MATERIALS AND METHODS: HCC samples were analyzed for MSI, MMR protein expression and coding microsatellite instability (cMSI). Since cMSI also affected SEC63 coding for an endoplasmic reticulum membrane protein with implications for intracellular protein translocation, its impact on hepatocyte growth control was assessed in an established short-term model. Recombinant inbred mouse lines (BXD) showing different basal SEC63 expression levels were treated with the chemocarcinogen diethylnitrosamine (DEN) intraperitoneally. Proliferation and apoptosis of hepatocytes were determined after 48 h using Ki67 and TUNEL assays. RESULTS: The HCC was high-grade microsatellite unstable with loss of MSH2 expression. cMSI was detected in four genes (ASTE1, SEC63, TAF1B, TGFBR2). However, only TGFBR2 is known to be involved in hepatocarcinogenesis. When investigating the impact of SEC63 expression on hepatocyte growth control in the murine model, low hepatic expression correlated significantly (p < 0.05) with a decrease in apoptosis and increased proliferative activity. CONCLUSIONS: For the first time, an HCC with characteristic molecular features of association with Lynch syndrome is described. The pro-carcinogenic growth behavior of hepatocytes with low SEC63 expression in the murine model indicates a potential role for SEC63 in hepatocarcinogenesis in general, but this needs further functional validation.

A frequent variant in the human bile salt export pump gene ABCB11 is associated with hepatitis C virus infection, but not liver stiffness in a German population.

BACKGROUND: The human ATP-binding cassette, subfamily B, member 11 (ABCB11) gene encodes the bile salt export pump, which is exclusively expressed at the canalicular membrane of hepatocytes. A frequent variant in the coding region, c.1331 T>C, leading to the amino acid exchange p.V444A, has been associated with altered serum bile salt levels in healthy individuals and predisposes homozygous carriers of the [C] allele for obstetric cholestasis. Recently, elevated bile salt levels were shown to be significantly associated with rates and risk of cirrhosis in patients with chronic hepatitis C virus (HCV) infection treated with pegylated interferon-α2 and ribavirin, suggesting a potential role for bile salt levels in HCV treatment outcomes and in the fibrogenic evolution of HCV-related liver disease. The aim of this study was to investigate a possible association of ABCB11 c.1331 T>C with hepatitis C virus (HCV) infection and fibrosis stages as assessed by non-invasive transient elastography in a German cohort of patients. METHODS: ABCB11 c.1331 T>C genotype was determined by allelic discrimination assay in 649 HCV infected cases and 413 controls. Overall, 444 cases were staged for fibrotic progression by measurement of liver stiffness. RESULTS: Homo- or heterozygous presence of the frequent [C] allele was associated with HCV positivity (OR = 1.41, CI = 1.02 - 1.95, p = 0.037). No association was detectable between the ABCB11 c.1331 T>C genotype and increased liver stiffness. CONCLUSIONS: Our data confirm that homozygous presence of the major [C] allele of ABCB11 c.1331 T>C is a genetic susceptibility factor for HCV infection, but not for liver fibrosis.

IL-13 Signaling in Liver Fibrogenesis.

Liver fibrosis is the final common pathway of chronic liver diseases irrespective of etiology. However, etiology deeply impacts progression and characteristics of liver fibrogenesis. IL-13 is the dominant pro-fibrotic cytokine in Schistosomiasis associated liver fibrogenesis. In vitro, IL-13 directly induces expression of fibrosis-associated genes, e.g., collagens or connective tissue growth factor, in hepatic stellate cells. Recently, potential effects of IL-13 in non-Schistosomiasis associated liver fibrosis have been uncovered. This review summarizes the potential roles of IL-13 in chronic liver disease of different etiologies, and the downstream events mediating IL-13 signaling in liver fibrogenesis.

Nuclear receptor variants in liver disease.

This review aims to provide a snapshot of the actual state of knowledge on genetic variants of nuclear receptors (NR) involved in regulating important aspects of liver metabolism. It recapitulates recent evidence for the application of NR in genetic diagnosis of monogenic ("Mendelian") liver disease and their use in clinical diagnosis. Genetic analysis of multifactorial liver diseases such as viral hepatitis or fatty liver disease identifies key players in disease predisposition and progression. Evidence from these analyses points towards a role of NR polymorphisms in common diseases, linking regulatory networks to complex and variable phenotypes. The new insights into NR variants also offer perspectives and cautionary advice for their use as handles towards diagnosis and treatment.

Decreased levels of active SMAD2 correlate with poor prognosis in gastric cancer.

BACKGROUND: TGF-ß plays a dual role in the progression of human cancer. During the early stages of carcinogenesis, TGF-ß functions as a tumor suppressor. During the late stages of tumor development, however, TGF-ß can promote tumor growth and metastasis. A shift in Smad2/3 phosphorylation from the carboxy terminus to linker sites is a key event determining biological function of TGF-ß in colorectal and hepatocellular carcinoma. In the present study, we investigated the potential role of differential Smad2/3 phosphorylation in gastric adenocarcinoma. METHODOLOGY: Immunohistochemical staining with anti-P-Smad2/3C and P-Smad2/3L antibodies was performed on 130 paraffin-embedded gastric adenocarcinoma specimens. The relationship between P-Smad2/3C and P-Smad2/3L immunohistochemical score and clinicopathologic characteristics of patients was analyzed. Real time PCR was used to measure mRNA expression of Smad2 and Smad3 in cancer and surrounding non-tumor tissue. PRINCIPAL FINDINGS: No significant P-Smad2L and/or P-Smad3L positive staining was detected in the majority of specimens (positive staining in 18/130 samples). Positive P-Smad2/3L staining was not associated with a decrease in carboxyterminal phosphorylation staining. Loss of P-Smad2C remarkably correlated with depth of tumor infiltration and poor differentiation of cancer cells in patients with gastric cancer. No correlation was detectable between P-Smad3C and clinicopathologic characteristics of gastric adenocarcinoma. However, co-staining analysis revealed that P-Smad3C co-localised with α-SMA and collagen I in gastric cancer cells, indicating a potential link between P-Smad3C and epithelial-to-mesenchymal transition of cancer. Real time PCR demonstrated reduced mRNA expression of Smad2 in gastric cancer when compared with surrounding non-tumor tissue in 15/16 patients. CONCLUSIONS: Loss of P-Smad2C tightly correlated with cancer invasion and poor differentiation in gastric cancer. Contrary to colorectal and hepatocellular carcinoma, canonical carboxy-terminal phosphorylation, but not linker phosphorylation, of Smad2 is critical for gastric cancer.

IL-13 induces connective tissue growth factor in rat hepatic stellate cells via TGF-ß-independent Smad signaling.

Connective tissue growth factor (CTGF) plays a central role in stimulating extracellular matrix deposition in the liver, and hence is considered a critical mediator of TGF-ß-dependent fibrogenesis. Hepatic stellate cells (HSCs) are known as the major source of CTGF in damaged liver. However, previous studies revealed that IL-13, rather than TGF-ß, represents the predominant inducer of CTGF expression in HSCs. We now dissected IL-13 downstream signaling that modulates CTGF expression in HSCs. IL-13 induces a time- and dosage-dependent increase of CTGF in a TGF-ß-independent manner. This process requires participation of different Smad proteins and their upstream receptor kinases (activin receptor-like kinases). Smad1 and Smad2 were identified as the key mediators of IL-13-dependent CTGF expression. Furthermore, IL-13 induces Stat6 phosphorylation in HSCs, but Stat6 was not involved in CTGF induction. Instead, the Erk1/2-MAPK pathway was found to be responsible for IL-13-induced early Smad phosphorylation and CTGF synthesis. We demonstrate that IL-13 induces CTGF expression in HSCs by activating TGF-ß-independent activin receptor-like kinase/Smad signaling via the Erk-MAPK pathway rather than via its canonical JAK/Stat6 pathway. These results provide an improved new insight into the molecular mechanisms of profibrotic IL-13 activities in the liver.

An update on genetic analysis of cholestatic liver diseases: digging deeper.

Investigations into the molecular mechanisms of cholestasis have revealed intricate and intriguing details of bile salt metabolism as well as its regulatory mechanisms in health and disease. Extensive studies on genotype-phenotype correlations in monogenic diseases, such as progressive familial and benign recurrent intrahepatic cholestasis, facilitate diagnostics and improve the risk assessment of hepatobiliary transporter gene variants in bile transport pathophysiology. While the comparatively easy targets in monogenic cholestasis have been identified for some time now, progress in complex liver disease is rather laborious but steady. Genome-wide association scans are the next step in gathering information about common contributors towards polygenic (multifactorial) cholestatic diseases. New determinants of bile salt metabolism affecting feedback loops within the liver or the enterohepatic circulation are presently under investigation for their contribution towards complex cholestatic syndromes.

Genome-wide association studies and genetic risk assessment of liver diseases.

Genetic tests can help clinicians to diagnose rare monogenic liver diseases. For most common liver diseases, however, multiple gene variants that have small to moderate individual phenotypic effects contribute to the overall risk of disease. An individual's level of risk depends on interactions between environmental factors and a wide range of modifier genes, which are yet to be identified systematically. The latest genome-wide association studies in large cohorts of patients with gallstones, fatty liver disease, viral hepatitis, chronic cholestatic liver diseases or drug-induced liver injury have provided new insights into the pathophysiology of these illnesses and have suggested the contribution of previously unsuspected pathogenic pathways. Studies in mouse models have identified further susceptibility genes for several complex liver diseases. As a result, in the future polygenic risk scores might help to define subgroups of patients at risk of developing liver diseases who would benefit from preventative measures and/or personalized therapy. Now that whole-genome sequencing is possible, comprehensive strategies for integrating genomic data and counseling of patients need to be developed.

The angiotensin-converting enzyme gene insertion-deletion polymorphism in a white British patient cohort with obstetric cholestasis.

The DD genotype of the angiotensin-converting enzyme (ACE) gene is over-represented in Finnish patients with obstetric cholestasis (OC). The purpose of this study was to establish whether this genotype is associated with cholestasis in UK cases. In a retrospective case-control study, we determined the ACE insertion/deletion frequencies in 166 British cases and 100 control women by polymerase chain reaction analysis. No significant difference in allele frequencies was found between these groups, but allele frequencies differed significantly between Finnish and UK OC cases (P = 0.0005). The prevalence of the DD genotype is lower in UK cases than in controls (χ(2) [1 d.f.] = 4.32, P = 0.05) and the odds ratio for OC associated with the DD genotypeis 0.54, 95% confidence interval 0.30-0.97. In contrast to Finnish OC cases, the DD genotype of the ACE is not increased in UK cases.

Functional variants of the central bile acid sensor FXR identified in intrahepatic cholestasis of pregnancy.

BACKGROUND AND AIMS: Intrahepatic cholestasis of pregnancy (ICP) is characterized by liver impairment, pruritus, and elevated maternal serum bile acids. It can cause premature delivery and intrauterine death. Bile acid synthesis, metabolism, and transport are regulated by the bile acid sensor FXR, and we hypothesized that genetic variation in FXR confers susceptibility to ICP. METHODS: The coding regions and intron/exon boundaries of FXR were sequenced in 92 British ICP cases of mixed ethnicity. Subsequently, a case-control study of allele frequencies of these variants in 2 independent cohorts of Caucasian ICP patients and controls was performed. Variants were cloned into an FXR expression plasmid and tested in functional assays. RESULTS: We identified 4 novel heterozygous FXR variants (-1g>t, M1V, W80R, M173T) in ICP. W80R was not present in Caucasians and M1V was detected uniquely in 1 British case. M173T and -1g>t occur both in Caucasian cases and controls, and we found a significant association of M173T with ICP (OR, 3.2; 95% confidence interval, 1.1-11.2; P = .02) when the allele frequencies of both Caucasian cohorts were analyzed together. We demonstrate functional defects in either translation efficiency or activity for 3 of the 4 variants (-1g>t, M1V, M173T). CONCLUSIONS: This is the first report of functional variants in FXR. We propose that these variants may predispose to ICP, and because FXR has a central role in regulating bile and lipid homeostasis they may be associated with other cholestatic and dyslipidemic disorders.

Absence of the genetic variant Val79Met in human chorionic gonadotropin-beta gene 5 in five European populations.

Chorionic gonadotropin (CG) is an essential signal in establishment and maintenance of pregnancy in humans and higher primates. A G-to-A transition in exon 3 of human CGbeta gene 5, changing the naturally occurring valine residue to methionine in codon 79 (Val(79)Met) has been reported at carrier frequency 4.2% in a random population from the Midwest of the United States. The biological activity of the variant hCG was similar to that of wild-type (WT) hCG. However, the Val(79)Met beta-subunit displayed impaired ability to assemble with alpha-subunit, and the amount of hCG alpha/beta heterodimers formed and secreted by transfected cells was seriously impaired in the previous study. Because of these functional implications we found it important to study the occurrence of the Val(79)Met hCGbeta variant in other populations. By using a PCR-RFLP method, a search for the Val(79)Met hCGbeta variant was carried out on a total of 580 DNA samples from five European populations (Finland, Denmark, Greece, Germany and the UK). The results demonstrated an absence of the polymorphism in these populations. Hence, the naturally occurring variant (Val(79)Met) of the hCGbeta gene 5, found previously at high frequency in the US, is clearly less common, or absent, in the European populations studied.

The 48 bp centromeric repeat is a functionally conserved motif in great apes and man showing protein-binding properties.

The centromere-kinetochore complex is a chromosomal assembly site including repeat motifs and protein binding properties thus mediating chromosome motility and mitotic regulation. Next to the alpha-satellite DNA family as well as human satellite III DNA, contribution of other repetitive sequences has to be strongly considered in centromere function. Here, we report the identification of centromeric 48 bp motifs, isolated from chimpanzee and orang-utan using an orthologous human DNA probe. Applying Southern hybridization we show that these sequences are restricted to hominoid species. Diminishing hybrid formation in gibbons suggested that the 48 bp repeat originated approximately 25-20 million years ago. Consistently, both chimpanzee as well as human repeat probes failed to generate any hybridization signal with the monkey species Cercopithecus aethiops and Aotes trivirgatus. Sequence deviations from the consensus of human repeat monomers of 6% and 10.4% in chimpanzee and orang-utan, respectively, were found within a 16 bp region of the 48 bp repeat units. Gel mobility shift assays using chimpanzee repeat dimers as probes revealed peptide-binding properties with human and chimpanzee nuclear extracts. Species-specific DNA-protein complexes remained unaffected by competition studies and indicated the presence of at least one novel interacting protein consisting of two subunits with 90 and 95 kDa. Our data suggest that the 48 bp repeat, next to alpha-satellite DNA, provides essential sequence information for specific DNA-protein interaction and they imply phylogenetic conservation of these binding properties in primates. The complex is likely involved in the proper formation and/or function of mammalian centromeres.