Sonja M. Kessler

IGF2 mRNA binding protein p62/IMP2-2 in hepatocellular carcinoma: antiapoptotic action is independent of IGF2/PI3K signaling

The insulin-like growth factor II (IGF2) mRNA binding protein (IMP) p62/IMP2-2, originally isolated from a hepatocellular carcinoma (HCC) patient, induces a steatotic phenotype when overexpressed in mouse livers. Still, p62 transgenic livers do not show liver cell damage but exhibit a pronounced induction of Igf2 and activation of the downstream survival kinase AKT. The aim of this study was to investigate the relation between p62 and IGF2 expression in the human system and to study potential antiapoptotic actions of p62. p62 and IGF2 mRNA levels were assessed by real-time RT-PCR. For knockdown and overexpression experiments, human hepatoma HepG2 and PLC/PRF/5 cells were transfected with siRNA or plasmid DNA. Phosphorylated AKT and ERK1/2 were analyzed by Western blot. Investigations of 32 human HCC tissues showed a strong correlation between p62 and IGF2 expression. Of note, p62 expression was increased markedly in patients with poor outcome. In hepatoma cells overexpression of p62 lowered levels of doxorubicin-induced caspase-3-like activity. Vice versa, knockdown of p62 resulted in increased doxorubicin-induced apoptosis. However, neither PI3K inhibitors nor a neutralizing IGF2 antibody showed any effects. Western blot analysis revealed increased levels of phosphorylated ERK1/2 in hepatoma cells overexpressing p62 and decreased levels in p62 knockdown experiments. When p62-overexpressing cells were treated with ERK1/2 inhibitors, the apoptosis-protecting effect of p62 was completely abrogated. Our data demonstrate that p62 exerts IGF2-independent antiapoptotic action, which is facilitated via phosphorylation of ERK1/2. Furthermore, p62 might serve as a new prognostic marker in HCC.

OVEREXPRESSION OF THE IGF2-mRNA BINDING PROTEIN p62 IN TRANSGENIC MICE INDUCES A STEATOTIC PHENOTYPE

BACKGROUND & AIMS The insulin-like growth-factor 2 (IGF2) mRNA binding protein p62 is highly expressed in hepatocellular carcinoma tissue. Still, its potential role in liver disease is largely unknown. In this study, we investigated pathophysiological implications of p62 overexpression in mice. METHODS We generated mice overexpressing p62 under a LAP-promotor. mRNA expression levels and stability were examined by real-time RT-PCR. Allele-specific expression of Igf2 and H19 was assessed after crossing mice with SD7 animals. The Igf2 downstream mediators pAKT and PTEN were determined by Western blot. RESULTS Hepatic p62 overexpression neither induced inflammatory processes nor liver damage. However, 2.5week old transgenic animals displayed a steatotic phenotype and improved glucose tolerance. p62 overexpression induced the expression of the imprinted genes Igf2 and H19 and their transcriptional regulator Aire (autoimmune regulator). Neither monoallelic expression nor mRNA stability of Igf2 and H19 was affected. Investigating Igf2 downstream signalling pathways showed increased AKT activation and attenuated PTEN expression. CONCLUSIONS The induction of a steatotic phenotype implies that p62 plays a role in hepatic pathophysiology.

Glucocorticoid-Induced Leucine Zipper: A Critical Factor in Macrophage Endotoxin Tolerance

Induction of glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids plays a key role in their anti-inflammatory action. In activated macrophages, GILZ levels are downregulated via tristetraprolin-mediated GILZ mRNA destabilization. To assess the functional significance of GILZ downregulation, we generated myeloid-specific GILZ knockout (KO) mice. GILZ-deficient macrophages displayed a higher responsiveness toward LPS, as indicated by increased TNF-α and IL-1β expression. This effect was due to an activation of ERK, which was significantly amplified in GILZ KO cells. The LPS-induced activation of macrophages is attenuated upon pretreatment of macrophages with low-dose LPS, an effect termed endotoxin tolerance. In LPS-tolerant macrophages, GILZ mRNA was stabilized, whereas ERK activation was strongly decreased. In contrast, GILZ KO macrophages exhibited a strongly reduced desensitization. To explore the contribution of GILZ expression in macrophages to endotoxin tolerance in vivo, we treated GILZ KO mice with repeated i.p. injections of low-dose LPS followed by treatment with high-dose LPS. LPS pretreatment resulted in reduced proinflammatory mediator expression upon high-dose LPS treatment in serum and tissues. In contrast, cytokine induction was preserved in tolerized GILZ KO animals. In summary, our data suggest that GILZ is a key regulator of macrophage functions.

Fatty Acid Elongation in Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma

Non-alcoholic steatohepatitis (NASH) represents a risk factor for the development of hepatocellular carcinoma (HCC) and is characterized by quantitative and qualitative changes in hepatic lipids. Since elongation of fatty acids from C16 to C18 has recently been reported to promote both hepatic lipid accumulation and inflammation we aimed to investigate whether a frequently used mouse NASH model reflects this clinically relevant feature and whether C16 to C18 elongation can be observed in HCC development. Feeding mice a methionine and choline deficient diet to model NASH not only increased total hepatic fatty acids and cholesterol, but also distinctly elevated the C18/C16 ratio, which was not changed in a model of simple steatosis (ob/ob mice). Depletion of Kupffer cells abrogated both quantitative and qualitative methionine-and-choline deficient (MCD)-induced alterations in hepatic lipids. Interestingly, mimicking inflammatory events in early hepatocarcinogenesis by diethylnitrosamine-induced carcinogenesis (48 h) increased hepatic lipids and the C18/C16 ratio. Analyses of human liver samples from patients with NASH or NASH-related HCC showed an elevated expression of the elongase ELOVL6, which is responsible for the elongation of C16 fatty acids. Taken together, our findings suggest a detrimental role of an altered fatty acid pattern in the progression of NASH-related liver disease.

Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation

OBJECTIVE Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. APPROACH AND RESULTS Degenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-κB activation after GILZ knockdown by siRNA and found that NF-κB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. CONCLUSION Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease.

The insulin-like growth factor 2 (IGF2) mRNA-binding protein p62/IGF2BP2-2 as a promoter of NAFLD and HCC?

Non-alcoholic fatty liver disease (NAFLD) represents the most common hepatic manifestation of chronic liver diseases in developed countries. Since non-alcoholic steatohepatitis (NASH) is responsible for a large proportion of cryptogenic cirrhosis and cirrhosis represents the main risk factor for hepatocellular carcinoma (HCC), HCC is a severe complication of end-stage NAFLD.1 Recent evidence published in this journal showed the therapeutic potential of an inhibition of the chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1) in NASH.2 The study by Baeck et al elegantly demonstrated that the pharmacological administration of an RNA oligonucleotide against MCP-1 ameliorates murine steatosis and inflammation. Since mice deficient of the MCP-1 receptor also showed attenuated fibrosis, MCP-1 was suggested as a critical link in the axis steatosis–inflammation–fibrosis.2 Here, we report that animals with a liver-specific overexpression of the insulin-like growth factor 2 ( IGF2 ) mRNA-binding protein p62/IMP2-2/IGF2BP2-2 exhibit distinctly elevated Ccl2 expression levels (figure 1A) when fed a methionine–choline-deficient (MCD) diet, which models all hepatic stages of NAFLD. Accordingly, in addition to elevated inflammatory gene expression, p62 transgenics had higher fat deposition (figure 1B) …

Susceptibility of Different Mouse Wild Type Strains to Develop Diet-Induced NAFLD/AFLD-Associated Liver Disease.

Although non-alcoholic and alcoholic fatty liver disease have been intensively studied, concerning pathophysiological mechanisms are still incompletely understood. This may be due to the use of different animal models and resulting model-associated variation. Therefore, this study aimed to compare three frequently used wild type mouse strains in their susceptibility to develop diet-induced features of non-alcoholic/alcoholic fatty liver disease. Fatty liver disease associated clinical, biochemical, and histological features in C57BL/6, CD-1, and 129Sv WT mice were induced by (i) high-fat diet feeding, (ii) ethanol feeding only, and (iii) the combination of high-fat diet and ethanol feeding. Hepatic and subcutaneous adipose lipid profiles were compared in CD-1 and 129Sv mice. Additionally hepatic fatty acid composition was determined in 129Sv mice. In C57BL/6 mice dietary regimens resulted in heterogeneous hepatic responses, ranging from pronounced steatosis and inflammation to a lack of any features of fatty liver disease. Liver-related serum biochemistry showed high deviations within the regimen groups. CD-1 mice did not exhibit significant changes in metabolic and liver markers and developed no significant steatosis or inflammation as a response to dietary regimens. Although 129Sv mice showed no weight gain, this strain achieved most consistent features of fatty liver disease, apparent from concentration alterations of liver-related serum biochemistry as well as moderate steatosis and inflammation as a result of all dietary regimens. Furthermore, the hepatic lipid profile as well as the fatty acid composition of 129Sv mice were considerably altered, upon feeding the different dietary regimens. Accordingly, diet-induced non-alcoholic/alcoholic fatty liver disease is most consistently promoted in 129Sv mice compared to C57BL/6 and CD-1 mice. As a conclusion, this study demonstrates the importance of genetic background of used mouse strains for modeling diet-induced non-alcoholic/alcoholic fatty liver disease.

Hepatic hepcidin expression is decreased in cirrhosis and HCC

et al. Expression of iron regulatory genes in a rat model of hepatocellular carcinoma. Liver Int 2006;26:976–985. [4] Youn P, Kim S, Ahn JH, Kim Y, Park JD, Ryu DY. Regulation of iron metabolism-related genes in diethylnitrosamine-induced mouse liver tumors. Toxicol Lett 2009;184:151–158. [5] Roessler S, Jia H-L, Budhu A, Forgues M, Ye Q-H, Lee J-S, et al. A unique metastasis gene signature enables prediction of tumor relapse in early-stage hepatocellular carcinoma patients. Cancer Res 2010;70:10202–10212. [6] Kijima H, Sawada T, Tomosugi N, Kubota K. Expression of hepcidin mRNA is uniformly suppressed in hepatocellular carcinoma. BMC Cancer 2008;8:167–175. [7] Girelli D, Pasino M, Goodnough JB, Nemeth E, Guido M, Castagna A, et al. Reduced serum hepcidin levels in patients with chronic hepatitis C. J Hepatol 2009;51:845–852. [8] Miura K, Taura K, Kodama Y, Schnabl B, Brenner DA. Hepatitis C virusinduced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity. Hepatology 2008;48:1420–1429. [9] Weizer-Stern O, Adamsky K, Margalit O, Ashur-Fabian O, Givol D, Amariglio N, et al. Hepcidin, a key regulator of iron metabolism, is transcriptionally activated by p53. Br J Haematol 2007;138:253–262. [10] Nault J-C, Zucman-Rossi J. Genetics of hepatocellular carcinoma: the next generation. J Hepatol 2014;60:224–226.

Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis

Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr–/–, a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2–/–), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr–/–;Mdr2–/– mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2–/– mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr–/–;Mdr2–/– mice had a pronounced down‐regulation of hepatoprotective genes Hnf6, Egfr, and Igf‐1, and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr–/–) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild‐type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr–/–;Mdr2–/– mice displayed a significant decrease in tumor incidence compared to Mdr2–/– mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. Conclusion: GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments. (Hepatology 2015;61:613‐626)

Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis

AIM To characterize how insulin-like growth factor 2 (IGF2) mRNA binding protein p62/IMP2-2 promotes steatohepatitis in the absence of dietary cholesterol. METHODS Non-alcoholic steatohepatitis (NASH) was induced in wild-type mice and in mice overexpressing p62 specifically in the liver by feeding the mice a methionine and choline deficient (MCD) diet for either two or four weeks. As a control, animals were fed a methionine and choline supplemented diet. Serum triglycerides, cholesterol, glucose, aspartate aminotransferase and alanine transaminase were determined by standard analytical techniques. Hepatic gene expression was determined by real-time reverse transcription-polymerase chain reaction. Generation of reactive oxygen species in liver tissue was quantified as thiobarbituric acid reactive substances using a photometric assay and malondialdehyde as a standard. Tissue fatty acid profiles and cholesterol levels were analyzed by gas chromatography-mass spectrometry after hydrolysis. Hepatocellular iron accumulation was determined by Prussian blue staining in paraffin-embedded formalin-fixed tissue. Filipin staining on frozen liver tissue was used to quantify hepatic free cholesterol levels. Additionally, nuclear localization of the nuclear factor kappa B (NF-κB) subunit p65 was examined in frozen tissues. RESULTS Liver-specific overexpression of the insulin-like growth factor 2 mRNA binding protein 2-2 (IGF2BP2-2/IMP2-2/p62) induces steatosis with regular chow and amplifies NASH-induced fibrosis in the MCD mouse model. Activation of NF-κB and expression of NF-κB target genes suggested an increased inflammatory response in p62 transgenic animals. Analysis of hepatic lipid composition revealed an elevation of monounsaturated fatty acids as well as increased hepatic cholesterol. Moreover, serum cholesterol was significantly elevated in p62 transgenic mice. Dietary cholesterol represents a critical factor for the development of NASH from hepatic steatosis. Filipin staining revealed increased free cholesterol in p62 transgenic livers, which were not diet-derived. The mRNA levels of the rate-limiting enzyme for cholesterol synthesis 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase or HMGCR) were not significantly upregulated, potentially due to increased cholesterol biosynthesis via elevated sterol regulatory element binding transcription factor 2 (SREBF2) gene expression and increased iron deposition in transgenic animals. CONCLUSION This study provides evidence that p62/IGF2BP2-2 drives the progression of NASH through elevation of hepatic iron deposition and increased production of hepatic free cholesterol.