Denk, Gerald Ulrich

Pusl, Thomas

Rust, Christian

Thasler, Wolfgang H.

Vennegeerts, Timo

Wimmer, Ralf

English

Universität München: Elektronischen Publikationen

Glycochenodeoxycholate-Induced Apoptosis Is Not Reduced by Augmenter of Liver Regeneration in the Human Hepatoma Cell Line HuH-7

Gerald Ulrich Denk

Ralf Wimmer

Timo Vennegeerts

Thomas Pusl

Wolfgang Thasler

Christian Rust

Crossref

Open Access LMU

Fax +41 61 306 12 34E-Mail karger@karger.chwww.karger.com Letter to the Editor  Digestion 2010;82:262–264  DOI: 10.1159/000297544  Glycochenodeoxycholate-Induced Apoptosis Is Not Reduced by Augmenter of Liver Regeneration in the Human Hepatoma Cell Line HuH-7  Gerald Ulrich Denk  a    Ralf Wimmer  a    Timo Vennegeerts  a    Thomas Pusl  c    Wolfgang Thasler  b    Christian Rust  a  Departments of  a   Medicine II and  b   Surgery – Grosshadern, University of Munich,  Munich , and c   First Medical Clinic, Klinikum Augsburg,  Augsburg , Germany a receptor for ALR, that may be important for signal transduction  [11] . Furthermore, ALR stimulates the mitogen-activated protein kinase cascade  [12] , decreases cy-tochrome P450, increases nuclear factor-  B activity  [13] , induces mRNA levels of pivotal enzymes of polyamine biosynthe-sis and enhances polyamine levels in hu-man hepatocytes  [14] . Although the beneficial effects of ALR in liver regeneration are well defined, a protective effect of ALR to reduce liver in-jury is less clear. Cholestasis is a common cause of liver disease and is characterized by an elevation of hepatic levels of hydro-phobic bile acids  [15–17] . Hepatocellular apoptosis induced by toxic, hydrophobic bile acids such as glycochenodeoxycholic acid (GCDCA) is a major cause of liver in-jury in cholestasis  [18–20] . In view of ALR’s beneficial effects on liver regeneration, we analyzed the role of ALR to reduce liver injury induced by GCDCA in an in vitro model. For this pur-pose, rat Na + -taurocholate cotransporting polypeptide (Ntcp) was stably transfected into the human hepatoma cell line HuH-7 with a pcDNA3.1 vector using the Dear Sir,  In contrast to other organs, the liver is characterized by an outstanding capacity to regenerate after injuries of diverse eti-ology. In this context, the protein aug-menter of liver regeneration (ALR) is con-sidered to be one of the major players in the process of hepatic regeneration  [1] and was found to support liver regenera-tion in partially hepatectomized rats, in dogs with Eck fistula (portocaval shunt)  [2] , in rats with thioacetamide-induced liver injury  [3] , and to decrease hepatic fi-brosis in carbon tetrachloride/ethanol-injured rats  [4] . Moreover, after partial hepatectomy, an increase of serum but not of liver ALR levels was observed in rats  [5] , and increased expression of ALR mRNA was observed in cirrhotic human livers  [6] . ALR is a homolog of the Erv1 protein from yeast that is essential for mi-tochondrial respiration and cell viability  [7] , and is a member of the Erv1/ALR pro-tein family of sulfhydryl oxidases  [8–10] . This protein family is important for mito-chondrial biogenesis, cell cycle, and he-patic and testicular organogenesis  [10] . Previous studies in rat hepatocytes and human HepG2 hepatoma cells described  Published online: June 24, 2010  Christian Rust, MD  Department of Medicine II – Grosshadern, University of Munich  Marchioninistrasse 15, DE–81377 Munich (Germany)  Tel. +49 89 70950, Fax +49 89 7095 8864  E-Mail christian.rust   @   med.uni-muenchen.de  © 2010 S. Karger AG, Basel0012–2823/10/0824–0262$26.00/0  Accessible online at:www.karger.com/dig FuGene  transfection reagent (Roche, Mannheim, Germany), similar as de-scribed previously  [21] . These Ntcp-trans-fected HuH-7 cells were then transiently transfected with a pcDNA3.1/Myc-HisB ALR construct  [22] using TransIT-LT1 re-agent (Mirus Bio LLC, Madison, Wisc., USA) for 24 h. This had no effect on cel-lular bile acid uptake as determined by liq-uid scintillation counting of Ntcp-HuH-7 cells after incubation with [ 3 H]taurocho-late (data not shown). As expected, incubation of Ntcp-HuH-7 cells with a low micromolar con-centration of the hydrophobic bile acid GCDCA (20   mol/l) for 4 h led to a 7-fold increase (p  ! 0.01) of apoptotic cell death as determined by activities of caspases 3 and 7 with the Apo-ONE homogeneous caspase-3/7 assay (Promega, Madison, Wisc., USA). Despite successful transfec-tion of Ntcp-HuH-7 cells with the hepato-poietic factor ALR as demonstrated by im-munoblotting against the Myc-tag (with a monoclonal mouse anti-Myc antibody from Invitrogen, Carlsbad, Calif., USA) and by PCR amplification of ALR mRNA (2,635-fold higher mRNA level after ALR  No Effect of ALR on GCDCA-Induced Apoptosis Digestion 2010;82:262–264 263transfection in comparison with controls), apoptotic cell death was not reduced under this condition ( fig. 1 ). In accordance with this observation, cell viability as deter-mined by formazan formation with the WST-1 assay (Roche) and by quantifica-tion of LDH activity  [23] in the culture me-dium was not affected by ALR transfection either (data not shown). Taken together, overexpression of ALR had no beneficial effect on apoptosis in-duced by low micromolar concentrations of GCDCA, which is one of the predomi-nant bile acids found in cholestatic pa-tients. Thus, our results suggest that ALR might not be beneficial in reducing hepa-tocellular apoptosis in human cholestatic liver disease. Acknowledgements  The pcDNA3.1/Myc-HisB plasmid con-taining human ALR was a generous gift from Dr. Yan Cao, Beijing Institute of Ra-diation Medicine (Beijing, China). Dr. Denk is a recipient of a research grant from the Else Kröner-Fresenius-Stiftung.  References  1 Pawlowski R, Jura J: ALR and liver regen-eration. Mol Cell Biochem 2006; 288: 159–169.  2 Francavilla A, Hagiya M, Porter KA, Polime-no L, Ihara I, Starzl TE: Augmenter of liver regeneration: its place in the universe of he-patic growth factors. Hepatology 1994; 20:  747–757.  3 Theocharis SE, Margeli AP, Agapitos EV, Mykoniatis MG, Kittas CN, Davaris PS: Ef-fect of hepatic stimulator substance admin-istration on tissue regeneration due to thio-acetamide-induced liver injury in rats. Scand J Gastroenterol 1998; 33: 656–663.  4 Manna Z, Guopei S, Minuk GY: Effects of he-patic stimulator substance, herbal medicine, selenium/vitamin E, and ciprofloxacin on cirrhosis in the rat. Gastroenterology 1996;  110: 1150–1155.  5 Gandhi CR, Kuddus R, Subbotin VM, Pre-lich J, Murase N, Rao AS, Nalesnik MA, Wat-kins SC, DeLeo A, Trucco M, Starzl TE: A fresh look at augmenter of liver regeneration in rats. Hepatology 1999; 29: 1435–1445.  6 Thasler WE, Schlott T, Thelen P, Heller-brand C, Bataille F, Lichtenauer M, Schlitt HJ, Jauch KW, Weiss TS: Expression of aug-menter of liver regeneration in human liver cirrhosis and carcinoma. Histopathology 2005; 47: 57–66.  7 Lisowsky T: Dual function of a new nuclear gene for oxidative phosphorylation and veg-etative growth in yeast. Mol Gen Genet 1992;  232: 58–64.  8 Lee JE, Hofhaus G, Lisowsky T: Erv1p from  Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase. FEBS Lett 2000; 477: 62–66.  9 Lisowsky T, Lee JE, Polimeno L, Francavilla A, Hofhaus G: Mammalian augmenter of liv-er regeneration protein is a sulfhydryl oxi-dase. Dig Liver Dis 2001; 33: 173–180.  10 Gatzidou E, Kouraklis G, Theocharis S: In-sights on augmenter of liver regeneration cloning and function. World J Gastroenterol 2006; 12: 4951–4958.  11 Wang G, Yang X, Zhang Y, Wang Q, Chen H, Wei H, Xing G, Xie L, Hu Z, Zhang C, Fang D, Wu C, He F: Identification and character-ization of receptor for mammalian hepato-poietin that is homologous to yeast ERV1. J Biol Chem 1999; 274: 11469–11472.  12 Li Y, Li M, Xing G, Hu Z, Wang Q, Dong C, Wei H, Fan G, Chen J, Yang X, Zhao S, Chen H, Guan K, Wu C, Zhang C, He F: Stimula-tion of the mitogen-activated protein kinase cascade and tyrosine phosphorylation of the epidermal growth factor receptor by hepato-poietin. J Biol Chem 2000; 275: 37443–37447. a Western blotpcDNA3.1pcDNA3.1-ALRpcDNA3.1pcDNA3.1-ALRMyc-tagPCRALRGAPDH02004006008001,0001,200Apoptosis(%ofcontrol)Control ALR GCDCA ALR +GCDCA**##b Fig. 1.  a A pcDNA3.1/Myc-HisB plasmid containing human ALR was successfully transfected into human hepatoma Ntcp-HuH-7 cells as demonstrated by immunoblotting using an antibody against the Myc-tag of the plasmid and by amplification of ALR mRNA by PCR.  b Transfection of ALR does not ameliorateGCDCA-induced apoptosis in Ntcp-HuH-7 cells that were incu-bated with or without the hydrophobic bile acid GCDCA (20   mol/l). Apoptosis was quantified by determination of the ac-tivities of the caspases 3 and 7. Results are expressed as % of con-trol and means  8 SD of each 7 independent experiments.  * *  p  ! 0.01 vs. control (no GCDCA);  ##  p  ! 0.01 vs. ALR (no GCDCA) (ANOVA with Tukey’s post-hoc test).  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Eur J Clin Invest 2002; 32: 437–442.  22 Cao Y, Fu YL, Yu M, Yue PB, Ge CH, Xu WX, Zhan YQ, Li CY, Li W, Wang XH, Wang ZD, Li YH, Yang XM: Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress. Free Radic Biol Med 2009;  47: 1057–1066.  23 Bergmeyer HU, Bernt E: Lactate dehydroge-nase; in Bergmeyer HU (ed): Methods ofEnzymatic Analysis. New York, Academic Press, 1983, vol 3, pp 574–579.  

AF: Cholestatic liver disease: pathophysiology and therapeutic options. Liver

ALR and liver regeneration.

Beuers U: Phosphatidylinositol 3-kinase-dependent signaling modulates taurochenodeoxycholic acid-induced liver injury and cholestasis in perfused rat livers.

CM: Modulation of hepatocyte apoptosis: cross-talk between bile acids and nuclear steroid receptors. Curr Med Chem

Davaris PS: Effect of hepatic stimulator substance administration on tissue regeneration due to thioacetamide-induced liver injury in rats.

Determination of bile acids in needle biopsies of human liver. Biochem Med

E: Lactate dehydrogenase; in Bergmeyer HU (ed): Methods of Enzymatic Analysis.

Everson G: Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids. Pediatr Res

Hofhaus G: Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase. Dig Liver Dis

Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1.

Lisowsky T: Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase.

Mechanism of cholestasis. 6. Bile acids in human livers with or without biliary obstruction. Gastroenterology

Minuk GY: Effects of hepatic stimulator substance, herbal medicine, selenium/vitamin E, and ciprofloxacin on cirrhosis in the rat. Gastroenterology

Starzl TE: A fresh look at augmenter of liver regeneration in rats. Hepatology

Starzl TE: Augmenter of liver regeneration: its place in the universe of hepatic growth factors. Hepatology

Stimulation of the mitogen-activated protein kinase cascade and tyrosine phosphorylation of the epidermal growth factor receptor by hepatopoietin.

T: Dual function of a new nuclear gene for oxidative phosphorylation and vegetative growth in yeast. Mol Gen Genet

Tauroursodeoxycholic acid mobilizes  -PKC after uptake in human HepG2 hepatoma cells.

Theocharis S: Insights on augmenter of liver regeneration cloning and function.

Weiss TS: Expression of augmenter of liver regeneration in human liver cirrhosis and carcinoma. Histopathology

Weiss TS: Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration.

Yang XM: Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress. Free Radic Biol Med

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Glycochenodeoxycholate-Induced Apoptosis Is Not Reduced by Augmenter of Liver Regeneration in the Human Hepatoma Cell Line HuH-7

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